Bendamustine derivatives and methods of using same

ABSTRACT

The present invention is directed to bendamustine esters and bendamustine amides and their use for the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/725,213, filed Nov. 12, 2012, and U.S. Provisional Application No.61/776,951, filed Mar. 12, 2013, the entireties of which areincorporated by reference herein.

TECHNICAL FIELD

The present invention is directed to esters and amides of bendamustinefor use in treating cancer.

BACKGROUND

Bendamustine,4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid:

is marketed as the hydrochloride salt under the trade names RIBOMUSTINand TREANDA and is a compound that has been used successfully for thetreatment of blood cancers such as chronic lymphocytic leukemia,Hodgkin's disease, non-Hodgkin's lymphoma, and multiple myeloma. Theseproducts are administered as intravenous infusions.

Use of bendamustine for the treatment of solid tumors is limited,however, by the compound's chemical instability in aqueous environment.Indeed, bendamustine has been reported as having a half-life of onlyabout 6-10 minutes in vivo. As a result, circulating levels ofbendamustine are not sustained for a long enough time for bendamustineto reach tumors outside of the circulatory system. Methods forincreasing the circulation time of bendamustine are needed.

SUMMARY

The present invention is directed to compounds of formula I:

wherein R₁ is C₆-C₂₄alkyl or polyethylene glycol; or pharmaceuticallyacceptable salt forms thereof. Methods of using compounds of formula Ifor the treatment of solid and non-solid cancer tumors are alsodescribed.

The invention is also directed to the use of compounds of formula IA:

wherein R is C₁-C₂₄alkyl or polyethylene glycol; or pharmaceuticallyacceptable salt forms thereof for the treatment of solid and non-solidcancer tumors.

The invention is further directed to compounds of formula II:

wherein R₂ is C₁-C₂₄alkylene; and R₃ is —COOC₁₋₃alkyl; or R₂-R₃ isC₁-C₂₄alkyl; or pharmaceutically acceptable salt forms thereof. Methodsof using compounds of formula II for the treatment of solid andnon-solid cancer tumors.

Nanoparticles including compounds of Formula I or IA, as well aslyophilized compositions comprising those nanoparticles, are also withinthe scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts plasma levels of certain embodiments of the invention inCD-1 mice, dosing at 3 mg/kg i.v. in 3% DMSO, 30% Solutol, 100 μL.

FIG. 2 depicts the effects of bendamustine hydrochloride and certainembodiments of the invention on tumor volumes of mice bearing MDA-MB-231xenografts.

FIG. 3 depicts the amount of bendamustine observed over time aftertreating MDA-MB-231 breast tumor S9 with certain embodiments of theinvention.

FIG. 4 depicts the amount of bendamustine observed over time aftertreating H460 non small cell lung tumor S9 with certain embodiments ofthe invention.

FIG. 5 depicts plasma levels of bendamustine in rats after dosing oneembodiment of the invention in rats using different formulations.

FIG. 6 depicts plasma levels of one embodiment of the invention in ratsafter dosing that embodiment in rats using different formulations.

FIG. 7 depicts Cryo-TEM images of nanoparticles of one embodiment of theinvention, bendamustine C₁₄ ester.

FIG. 8 depicts plasma levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 9 depicts blood levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 10 depicts brain levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 11 depicts liver levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 12 depicts lung levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 13 depicts spleen levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 14 depicts kidney levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 15 depicts plasma, blood, and organ levels of bendamustine in ratafter administration of one embodiment of the invention, bendamustineC₁₂ ester (as liquid formulation), after dosing rats at 30 mg/kg i.v., 1mL/kg.

FIG. 16 depicts plasma, blood, and organ levels of one embodiment of theinvention, bendamustine C₁₂ ester, in rat after dosing rats at 30 mg/kgi.v., 1 mL/kg with bendamustine C₁₂ ester (as liquid formulation).

FIG. 17 depicts plasma, blood, and organ levels of bendamustine in ratafter administration of one embodiment of the invention, bendamustineC₁₂ ester (as nanoparticle formulation), after dosing rats at 30 mg/kgi.v., 1 mL/kg.

FIG. 18 depicts plasma, blood, and organ levels of one embodiment of theinvention, bendamustine C₁₂ ester, in rat after dosing rats at 30 mg/kgi.v., 1 mL/kg with bendamustine C₁₂ ester (as nanoparticle formulation).

FIG. 19 depicts plasma levels of bendamustine in rat afteradministration of embodiments of the invention, bendamustine PEG-2000ester and bendamustine PEG-5000 ester, after dosing rats at 3 mg-eq/kgi.v., 1 mL/kg. Comparison is with TREANDA.

FIG. 20 depicts plasma levels of bendamustine in CD-1 mice after dosingembodiments of the invention at 3 mg/kg i.v., 3% DMSO, 30% Solutol.

FIG. 21 depicts a representative nanoparticle embodiment of theinvention at a magnification of 52,000×. Observed in the sample are:spherical particles that appear evenly denser than the surroundingbuffer (left-most arrows), small particles in the background (right-mostarrow). Insets show the two particles denoted by the left-most arrows ata larger scale. Distance between crosses in the left image is 28 nm,between crosses in the right inset is 43 nm. Scale Bar: 200 nm.

FIG. 22 depicts a representative nanoparticle embodiment of theinvention at a magnification of 52,000×. Observed in the sample are:spherical particles that appear evenly denser than the surroundingbuffer (left-most arrows), small particles in the background (right-mostarrow). Insets show the two particles denoted by the left-most arrows ata larger scale. Distance between crosses in the left image is 28 nm,between crosses in the right inset is 43 nm. Scale Bar: 200 nm.

FIG. 23 depicts tumor volumes following administration of VELCADE®,bendamustine, and bendamustine C12 ester nanoparticles.

FIG. 24 depicts body weight measurements following administration ofVELCADE®, bendamustine, and bendamustine C12 ester nanoparticles.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

It has been discovered that converting the carboxylic acid moiety ofbendamustine to a C₁-C₂₄alkyl ester group, a polyethylene glycol estergroup, or a C₁-C₂₄alkyl amide group results in compounds that providelonger circulating times for bendamustine. While not wishing to be boundto any particular theory, it is presumed that the ester or amide moietyreduces the solubility of the bendamustine molecule, resulting in aprotective effect against the aqueous environment. Over time, the esteror amide moiety is hydrolyzed to reveal the carboxylic acid moiety ofthe active bendamustine molecule. The overall result is thatbendamustine is generated over time.

By varying the number of carbons in the ester/amide moiety, thelipophilicity of the resulting bendamustine derivative can be modified.Increasing lipophilicity has been correlated to increased stability ofthe ester/amide and longer circulating times of bendamustine.

Within the scope of the invention are compounds of formula IA:

wherein R is C₁-C₂₄alkyl or polyethylene glycol; or a pharmaceuticallyacceptable salt form thereof. Compounds of formula IA are useful for thetreatment of solid or non-solid cancer tumors in patients.

Compounds of the invention can be formulated into pharmaceuticalcompositions comprising the compound of formula IA, or apharmaceutically acceptable salt form thereof, and a pharmaceuticallyacceptable carrier or diluent. In preferred pharmaceutical compositionsof the invention, R is C₁₀-C₂₄alkyl. Preferably, R is C₁₀alkyl. Alsopreferred is where R is C₁₂alkyl. Other preferred embodiments includethose where R is C₁₄alkyl. Compositions where R is C₁₆alkyl are alsopreferred.

Other embodiments of the invention include nanoparticles comprising acompound of formula IA.

Also within the scope of the invention are methods of treating solid ornon-solid cancer tumors in patients comprising administering to thepatient a compound of formula IA. Preferred solid or non-solid tumorsinclude chronic lymphocytic leukemia, Hodgkin's disease, indolentnon-Hodgkin's lymphoma (T-cell lymphoma, B-cell lymphoma), aggressivenon-Hodgkin's lymphoma, multiple myeloma, acute lymphocytic leukemia,breast cancer or lung cancer (small cell lung cancer (SCLC) andnon-small cell lung cancer (NSCLC), for example). Other solid andnon-solid cancer tumors are also envisioned as being treatable withcompounds and compositions of the invention, such as for example,sarcoma, bladder cancer, cervical cancer, testicular cancer, melanoma,glioblastoma, colon cancer, head and neck cancer, ovarian cancer, andprostate cancer. Other solid and non-solid cancer tumors are alsoenvisioned as being treatable with compounds of the invention, forexample, breast cancer, pancreatic cancer, and gastric cancer.

Preferred compounds of the invention are those of formula I:

wherein R₁ is C₆-C₂₄alkyl or polyethylene glycol; or a pharmaceuticallyacceptable salt form thereof. Compounds of formula I are useful for thetreatment of solid or non-solid cancer tumors in patients.

In preferred embodiments, R₁ is C₈-C₂₄alkyl. In other embodiments, R₁ isC₁₀-C₂₄alkyl. In yet other embodiments, R₁ is C₁₂-C₂₄alkyl. In stillother embodiments, R₁ is C₁₄-C₂₄alkyl. Also preferred are thosecompounds of formula I wherein R₁ is C₁₆-C₂₄alkyl. In other embodiments,R₁ is C₁₈-C₂₄alkyl.

In other embodiments, R₁ is C₁₀alkyl. In yet other embodiments, R₁ isC₁₂alkyl. In still other embodiments, R₁ is C₁₄alkyl. In otherembodiments, R₁ is C₁₆alkyl.

Also within the scope of the invention are pharmaceutical compositionscomprising a compound of formula I and a pharmaceutically acceptablecarrier or diluent.

Other embodiments of the invention include nanoparticles comprising acompound of formula I.

Also within the scope of the invention are methods of treating cancercomprising administering to a patient a compound of formula I. A numberof cancers, including those that involve solid tumors as well as thosethat do not involve solid tumors may be amenable to such treatment.These cancers include chronic lymphocytic leukemia, Hodgkin's disease,indolent non-Hodgkin's lymphoma (T-cell lymphoma, B-cell lymphoma),aggressive non-Hodgkin's lymphoma, multiple myeloma, acute lymphocyticleukemia, breast cancer or lung cancer (small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), for example). Additional cancersthat are also envisioned as being treatable with compounds andcompositions of the invention are those characterized by the presence ofsolid tumors, include sarcoma, bladder cancer, cervical cancer,testicular cancer, melanoma, glioblastoma, colon cancer, head and neckcancer, ovarian cancer, and prostate cancer. Other solid and non-solidcancer tumors are also envisioned as being treatable with compounds ofthe invention, for example, breast cancer, pancreatic cancer, andgastric cancer.

Particularly preferred compounds of the invention include:

Also within the scope of the invention are compounds of formula II:

wherein R₂ is C₁-C₂₄alkylene; and R₃ is —COOC₁₋₃alkyl; or R₂-R₃ isC₁-C₂₄alkyl; or a pharmaceutically acceptable salt form thereof.Compounds of formula II are useful for the treatment of solid ornon-solid cancer tumors in patients.

In preferred embodiments of compounds of formula II, R₂-R₃ isC₈-C₂₄alkyl. In other embodiments, R₂-R₃ is C₁₀-C₂₄alkyl. In still otherembodiments, R₂-R₃ is C₁₂-C₂₄alkyl. In yet other embodiments, R₂-R₃ isC₁₄-C₂₄alkyl. Also preferred is when R₂-R₃ is C₁₆-C₂₄alkyl. In otherembodiments, R₂-R₃ is C₁₈-C₂₄alkyl.

Preferably, for compounds of formula II, R₂-R₃ is C₁₀alkyl. Alsopreferred is when R₂-R₃ is C₁₂alkyl. In other embodiments, R₂-R₃ isC₁₄alkyl. In yet other embodiments, R₂-R₃ is C₁₆alkyl.

In other embodiments, R₂ is C₂alkylene and R₃ is —COOCH₃.

Preferred compounds of formula II include:

Also within the scope of the invention are pharmaceutical compositionscomprising a compound of formula II and a pharmaceutically acceptablecarrier or diluent.

Other embodiments of the invention include nanoparticles comprising acompound of formula II.

In one embodiment of the invention, the compounds and compositions ofthe invention are used to treat patients who are resistant to one ormore chemotherapeutic agents, such as, for example, alkylating agents.Exemplary alkylating agents to which patients may be resistant include:nitrogen mustards; ethylenimes; alkylsulfonates; triazenes; piperazines;and nitrosureas. More specific examples of the various types ofchemotherapeutic agents to which patients can become resistant arelisted below. Patients resistant to one or more of these agents wouldbenefit by treatment with the compounds and compositions of theinvention.

Nitrogen Mustards

Mechlorethamine, marketed under the trade name Mustargen®, is given byinjection to treat Hodgkin's disease and non-Hodgkin's lymphoma, and asa palliative therapy for breast and lung cancers, and given as a topicaltreatment for skin lesions of mycosis fungoides (cutaneous T-celllymphoma).

Ifosfamide, sold under the trade name Ifex®, is used to treat bothHodgkin's and non-Hodgkin's lymphoma, as well as recurrent testicularcancer and germ cell tumors, sarcomas, lung cancer, bladder cancer, headand neck cancer, and cervical cancer.

Melphalan is a chemotherapy drug sold under the brand name Alkeran®, andis also referred to as L-PAM or phenylalanine mustard. It is used totreat multiple myeloma, ovarian cancer, neuroblastoma, rhabdomyosarcoma,and breast cancer.

Chlorambucil is sold by the trade name Leukeran®, and is most widelyused to treat chronic lymphocytic leukemia, malignant lymphomasincluding lymphosarcoma, giant follicular lymphoma, and Hodgkin'sdisease. It has also been successfully used to treat non-Hodgkin'slymphoma, breast, ovarian and testicular cancer, Waldenstrom'smacroglobulinemia, thrombocythemia, and choriocarcinoma.

Cyclophosphamide is marketed as Cytoxan® or Neosar®, and is used totreat Hodgkin's and non-Hodgkin's lymphoma, Burkitt's lymphoma, chroniclymphocytic leukemia, chronic myelocytic leukemia, acute myelocyticleukemia, acute lymphocytic leukemia, t-cell lymphoma, multiple myeloma,neuroblastoma, retinoblastoma, rhabdomyosarcoma, Ewing's sarcoma;breast, testicular, endometrial, ovarian, and lung cancers.

Nitrosoureas

Streptozocin is sold under the trade name Zanosar®, and is used to treatislet cell pancreatic cancer.

Carmustine is also known as BiCNU® or BCNU, and is used for some kindsof brain tumors, glioblastoma, brainstem glioma, medulloblastoma,astrocytoma, ependymoma, and metastatic brain tumors. It is also used intreatment for multiple myeloma, Hodgkin's disease, non-Hodgkin'slymphoma, melanoma, lung cancer, and colon cancer.

Lomustine, also known as CCNU or CeeNU®, is used to treat primary andmetastatic brain tumors, Hodgkin's disease and non-Hodgkin's lymphoma,and has also been used for melanoma, lung, and colon cancer.

Alkyl Sulfonates

Busulfan, sold under trade names Busulfex® and Myleran®, is used totreat chronic myelogenous leukemia.

Triazines

Dacarbazine is sold under the trade name DTIC-Dome® and is used to treatmetastatic malignant melanoma, Hodgkin's disease, soft tissue sarcomas,neuroblastoma, fibrosarcomas, rhabdomyosarcoma, islet cell carcinoma,and medullary thyroid carcinoma.

Temozolomide is sold under the trade name Temodar®, and is used to treatthe specific types of brain tumors anaplastic astrocytoma andglioblastoma multiforme.

Ethylenimines

Thiotepa, known under the trade name Thioplex®, is an alkylating agentused to treat breast cancer, ovarian cancer, Hodgkin's disease, andnon-Hodgkin's lymphoma.

Altretamine is sold under the trade name Hexalen®, and is also calledhexamethylmelamine or HMM. It is used to treat ovarian cancer.

As used herein, “C₁-C₂₄alkyl” refers to straight or branched, saturatedhydrocarbon groups containing from one to 24 carbon atoms.Representative alkyl groups include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,n-pentyl, n-hexyl, n-heptyl, n-octyl, n-dodecyl, etc. Within the scopeof the invention, “C₁-C₂₄alkyl” also encompasses “cycloalkyl,” whichrefers to monocyclic, bicyclic, and tricyclic saturated hydrocarbons,for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclobutyl,bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptyl, adamantyl, and the like.

As used herein “polyethylene glycol,” also referred to as “PEG,” refersto polymers of the general formula H(OCH₂CH₂)_(n)O— orH(OCH₂CH₂)_(n)OCH₃, wherein n is at least 4. The preferred PEG has anaverage molecular weigh of from about 200 to about 5000 Daltons, with amore preferred PEG from about 2000 to about 5000 Daltons.

As used herein, “pharmaceutically acceptable carrier or diluent” refersto solvents, dispersion media, coatings, bulking agents, stabilizingagents, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanol,sugars such as trehalose and sucrose, polyalcohols such as mannitol,sorbitol, mixtures of sugars and polyalcohols, and sodium chloride.Pharmaceutically acceptable carriers may further include auxiliarysubstances such as wetting or emulsifying agents, preservatives, andbuffers.

As used herein, “pharmaceutical composition” refers to a compositionsuitable for administration in medical or veterinary use. Such compoundswill preferably include a compound of the invention in combination withone or more carriers and/or diluents. Such compositions are alsoreferred to as “formulations.”

As used herein, “administering” refers to any means within the art bywhich compounds of the invention can be delivered to the patient.Preferred administration methods include local administration, that is,administration of the compounds of the invention directly to thelocation where the effect of the compounds is desired, and systemicadministration. Examples of administration methods include, but are notlimited to, oral, enteric, sublingual, sublavial, subcutaneous, nasal,intravenous, intraarterial, intramuscular, and intraperitonealadministration.

As used herein, “solid tumor” refers to a malignant tumor that is alocalized mass of tissue. Examples of solid cancer tumors includelymphomas, sarcomas, and carcinomas and include breast cancer, braincancer, bone cancer, colon cancer, pancreatic cancer, lung cancer, andthe like.

As used herein, a “non-solid tumor cancer” refers most commonly tohematologic cancers, that is, malignant cancers of the blood. Examplesof non-solid tumor cancers include chronic lymphocytic leukemia,Hodgkin's disease, indolent non-Hodgkin's lymphoma (T-cell lymphoma,B-cell lymphoma), multiple myeloma, and the like.

As used herein, “nanoparticles” refers to a particle having an averagediameter of about 0.2 μm or less, preferably about 0.1 μm or less, asmeasured by Malvern Zetasizer.

EXPERIMENTAL SECTION Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid methyl ester (bendamustine C₁ ester)

Method A: To a 1 L three neck, round bottom flask equipped with anoverhead stirrer, condenser with nitrogen sweep, and thermocouple withtemperature controller was charged4-(5-amino-1-methyl-1H-benzoimidazol-2-yl)-butyric acid methyl ester(10.2 g, 41.2 mmol 1.0 eq), and chloroacetic acid (81.9 g, 866 mmol),and 20 mL of dry tetrahydrofuran (THF). The slurry was stirred in a tapwater bath to allow all of the solids to be dissolved. Borane-THF (288mL, 288 mmol) was added slowly via an additional funnel over 25 minutes.When the addition of BH₃-THF was complete, the resulting reactionsolution was stirred at room temperature for 1.5 hours and then heatedat 58° C. using a heat mantle for 45 minutes. The reaction was cooledand held at room temperature overnight and then quenched with methanol(10 mL). The resulting solution was concentrated to approximatelyone-third weight by evaporation on the rotary evaporator and neutralizedto pH 8-9 with an aqueous solution of sodium hydroxide in an ice-waterbath. The solid was collected by vacuum filtration, washed with water(200 mL), then reslurried with a dilute aqueous solution of sodiumbicarbonate (50 mL) for 20 minutes. Filtration was followed by dryingwith house vacuum at room temperature overnight, giving a tan solid (9.6g, 63% yield, 93 A % purity) ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.8Hz, 1H), 6.92 (d, J=2.3 Hz, 1H), 6.78 (dd, J=8.8, 2.3 Hz, 1H), 3.70 (brs, 8H), 3.66 (s, 3H), 3.59 (s, 3H), 2.83 (t, J=7.4 Hz, 2H), 2.48 (t,J=7.4 Hz, 2H, overlapped partially with DMSO), 2.01 (quint, J=7.4 Hz,2H); LC/MS (ESI, m/z) 372 (M+1), mp 60-63° C. dec.

Method B: To a 2 L three-neck glass vessel equipped with a heatingmantle, thermocouple, condenser, nitrogen inlet/outlet, and overheadstirrer was charged bendamustine HCl (50.0 g, 126.7 mmol, 1.0 eq.),methanol (500 mL), and methanesulfonic acid (2.47 mL, 38.1 mmol). Thereaction mixture was heated to reflux and stirred at 65° C. for onehour. The reaction solution was cooled to 40° C. and concentrated undervacuum. Water (500 mL) was added to the concentrated residue, and asaturated aqueous solution of NaHCO₃ (150 mL) was used to neutralize themixture to pH 6 over 1.5 hours. The product was collected by filtration,washed with water (150 mL) and dried at 40° C. under vacuum, giving awhite, powdery solid, 44.2 g (94% yield) with 98.4 A % purity by HPLC.

Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid ethyl ester (bendamustine C₂ ester)

Method A: To a 1 L three-neck glass vessel equipped with a heatingmantle, thermocouple, condenser, nitrogen inlet/outlet, and overheadstirrer was charged bendamustine HCl (30.0 g, 76 mmol, 1.0 eq.), ethanol(300 mL), and methanesulfonic acid (1.48 mL, 22.8 mmol). The reactionmixture was heated at 70° C. for one hour. The reaction solution wascooled to 40° C. and concentrated under vacuum. Water (300 mL) was addedto the concentrated residue, and a saturated aqueous solution of NaHCO₃(115 mL) was used to neutralize the mixture to pH 6 over 1.5 hours. Theproduct was collected by filtration, washed with water (100 mL) anddried at 40° C. under vacuum, giving a white solid, 28.6 g (97% yield)with 99.2 A % purity by HPLC. ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.8Hz, 1H), 6.92 (d, J=2.3 Hz, 1H), 6.78 (dd, J=8.8, 2.3 Hz, 1H), 4.04(quint, J=7.12 Hz, 2H), 3.70 (br s, 8H), 3.66 (s, 3H), 2.83 (t, J=7.4Hz, 2H), 2.45 (t, J=7.4 Hz, 2H, overlapped partially with DMSO), 2.00(quint, J=7.4 Hz, 2H), 1.18 (t, J=7.12 Hz, 3H).

Method B: To a 500 mL three-neck glass flask equipped with a heatingmantle, thermocouple, condenser, nitrogen inlet/outlet, and overheadstirrer was charged 4-(5-amino-1-methyl-1H-benimidazol-2-yl)-butyricacid ethyl ester (6.4 g, 1.0 eq.), chloroacetic acid (42.5 g), andtetrahydrofuran (THF, 13 mL). The resulting mixture was stirred for 1.5hours in a water bath at room temperature. Borane-THF (150 mL) was addedover 20 minutes. Once the charge was complete, the reaction mixture washeated to 55-58° C. and stirred for 1.5 hours. In-process analysis byHPLC showed 94 A % of the desired product. The reaction was cooled toroom temperature and telescoped to the next step of hydrolysis togenerate bendamustine.

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid butyl ester (bendamustine C₄ ester)

A 250 mL three neck round bottom flask was equipped with an overheadstirrer, thermocouple, temperature controller and nitrogen sweep thencharged with 10.0 g (25.34 mmol) of bendamustine hydrochloride, 1.9 g(2.35 mL, 25.6 mmol, 1.01 eq) of 1-butanol, 5.3 g (25.6 mmol, 1.01 eq)of dicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31 g (2.54 mmol,0.1 eq) of DMAP. The reaction was stirred at room temperature overnightat which time an in process analysis indicated the reaction wascomplete. Solids were removed by vacuum filtration and washed with 25 mLof MDC. The filtrate was washed with saturated aqueous sodiumbicarbonate solution (2×100 mL), DI water (1×100 mL) and brine (1×100mL) before drying over sodium sulfate, filtering and concentrating todryness in vacuo to a brown oil. The oil was triturated with 25 mL ofMDC and the solid impurities were removed by vacuum filtration andwashed with 25 mL of MDC. The filtrate was concentrated to dryness invacuo to yield 9.5 g (22.8 mmol, 90%) of the product as a clear lightbrown oil with an HPLC purity of 94.5 A %. ¹H NMR (400 MHz, CDCl₃) δ7.17 (d, J=8.76 Hz, 1H), 7.08 (d, J=2.32 Hz, 1H), 6.77 (dd, J=2.36, 8.8Hz, 1H), 4.05 (t, J=6.76 Hz, 2H), 3.72 (m, 4H), 3.69 (s, 3H), 3.63 (m,4H), 2.91 (t, J=7.4 Hz, 2H), 2.49 (t, J=7.1 Hz, 2H), 2.18 (m, 2H), 1.60(m, 2H), 1.32 (m, 2H), 0.89 (t, J=4.56 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid hexyl ester (bendamustine C₆ ester)

Method A: A 250 mL three neck round bottom flask was equipped with anoverhead stirrer, thermocouple, temperature controller and nitrogensweep then charged with 10.0 g (25.34 mmol) of bendamustinehydrochloride, 2.62 g (3.22 mL, 25.6 mmol, 1.01 eq) of 1-hexanol, 5.3 g(25.6 mmol, 1.01 eq) of dicyclohexylcarbodiimide (DCC), 100 mL of MDCand 0.31 g (2.54 mmol, 0.1 eq) of DMAP. The reaction was stirred at roomtemperature overnight at which time an in process analysis indicated thereaction was complete. Solids were removed by vacuum filtration andwashed with 25 mL of MDC. The filtrate was washed with saturated aqueoussodium bicarbonate solution (2×100 mL), DI water (1×100 mL) and brine(1×100 mL) before drying over sodium sulfate, filtering andconcentrating to dryness in vacuo to a brown oil. The oil was trituratedwith 25 mL of MDC and the solid impurities were removed by vacuumfiltration and washed with 25 mL of MDC. The filtrate was concentratedto dryness in vacuo to yield 8.91 g (20.1 mmol, 79%) of the product as aclear light brown oil with an HPLC purity of 91.9 A %. ¹H NMR (400 MHz,CDCl₃) δ 7.17 (d, J=8.76 Hz, 1H), 7.08 (d, J=2.32 Hz, 1H), 6.77 (dd,J=2.36, 8.8 Hz, 1H), 4.05 (t, J=6.76 Hz, 2H), 3.72 (m, 4H), 3.69 (s,3H), 3.63 (m, 4H), 2.91 (t, J=7.4 Hz, 2H), 2.49 (t, J=7.1 Hz, 2H), 2.18(m, 2H), 1.60 (m, 2H), 1.32 (m, 6H), 0.89 (t, J=4.56 Hz, 3H).

Method B: A one liter 4-necked round bottom flask equipped with anoverhead stirrer, thermocouple and nitrogen in/oulet was charged with 30g (76.0 mmol) of bendamustine hydrochloride and 300 mL ofdichloromethane. Agitation was begun and 10.6 mL (7.69 g, 76.0 mmol) oftriethlamine was added via syringe then stirred for 15 minutes at roomtemperature before adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDAC, 21.86 g, 114 mmol) and n-hexyl alcohol (9.57 mL, 7.84 g, 76.8mmol). The cloudiy white reaction mixture became a clear solution afterstirring for 20 minutes. Agitation was continued for 22.5 h at roomtemperature and 30° C. for one hour until reaction was complete by HPLCanalysis. DI water (300 mL) was charged to quench the reaction and thepH was adjusted to 6 using 1N HCl. The layers were separated, aqueouswas extracted with dichloromethane (100 mL), before combining theorganic phases and drying over sodium sulfate. After filtration andconcentration to dryness under vacuum a clear yellow oil was obtainedwhich was purified by column chromatography (25 to 50% ethyl acetate inheptane). A total of 16.5 g (37.3 mmol, 49.1%) was recovered as a thickyellow oil with 99.1 A % purity by HPLC.

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyric aidoctyl ester (bendamustine C₈ ester)

A 250 mL three neck round bottom flask was equipped with an overheadstirrer, thermocouple, temperature controller and nitrogen sweep thencharged with 10.0 g (25.34 mmol) of bendamustine hydrochloride, 3.33 g(4.03 mL, 25.6 mmol, 1.01 eq) of 1-octanol, 5.3 g (25.6 mmol, 1.01 eq)of dicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31 g (2.54 mmol,0.1 eq) of DMAP. The reaction was stirred at room temperature overnightat which time an in process analysis indicated the reaction wascomplete. Solids were removed by vacuum filtration and washed with 25 mLof MDC. The filtrate was washed with saturated aqueous sodiumbicarbonate solution (2×100 mL), DI water (1×100 mL) and brine (1×100mL) before drying over sodium sulfate, filtering and concentrating todryness in vacuo to a brown oil. The oil was triturated with 25 mL ofMDC and the solid impurities were removed by vacuum filtration andwashed with 5 mL of MDC. The filtrate was concentrated to dryness invacuo to yield 9.7 g (20.5 mmol, 81%) of the product as a clear lightbrown oil with an HPLC purity of 91.9 A %. ¹H NMR (400 MHz, CDCl₃) δ7.17 (d, J=8.76 Hz, 1H), 7.08 (d, J=2.28 Hz, 1H), 6.77 (dd, J=2.4, 8.76Hz, 1H), 4.05 (t, J=6.8 Hz, 2H), 3.72 (m, 4H), 3.69 (s, 3H), 3.63 (m,4H), 2.91 (t, J=7.44 Hz, 2H), 2.49 (t, J=7.12 Hz, 2H), 2.18 (m, 2H),1.60 (m, 2H), 1.32 (m, 10H), 0.89 (t, J=6.72 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid decyl ester (bendamustine C₁₀ ester)

Method A: A 250 mL three necked round bottom flask equipped with a stirbar, thermocouple and nitrogen in/outlet was charged with 10.0 g (25.3mmol) of bendamustine hydrochloride, 4.9 mL (4.08 g, 25.6 mmol, 1.01 eq)of decyl alcohol, 5.3 g (25.6 mmol, 1.01 eq) of dicyclohexylcarbodiimide (DCC), 100 mL of dichloromethane and 0.31 g (2.53 mmol, 0.1eq) of N,N-dimethylamino pyridine (DMAP). The reaction mixture wasstirred at room temperature for 18 hours at which time an HPLC analysisindicated the reaction was complete. Solids were removed by vacuumfiltration and the filtrate was washed with DI water (2×100 mL) andsaturated sodium bicarbonate (1×100 mL) before being dried over sodiumsulfate. The organic phase was filtered to remove the drying agent thenconcentrated to dryness in vacuo to give 9.6 g (19.2 mmol, 75.9%) of thedesired product as a low melting white solid with an HPLC purity of 94.1A %. ¹H NMR (400 MHz, CDCl₃) δ 7.17 (d, J=8.76 Hz, 1H), 7.08 (d, J=2.32Hz, 1H), 6.77 (dd, J=2.4, 8.76 Hz, 1H), 4.05 (t, J=6.76 Hz, 2H), 3.72(m, 4H), 3.69 (s, 3H), 3.63 (m, 4H), 2.91 (t, J=7.4 Hz, 2H), 2.49 (t,J=7.08 Hz, 2H), 2.18 (m, 2H), 1.60 (m, 2H), 1.32 (m, 14H), 0.87 (t,J=6.68 Hz, 3H).

Method B: A 250 mL 4-necked round bottom flask equipped with a magneticstir bar, thermocouple and nitrogen in/oulet was charged with 10 g (25.3mmol) of bendamustine hydrochloride and 100 mL of dichloromethane.Agitation was begun and 3.53 mL (2.56 g, 25.3 mmol) of triethlamine wasadded via syringe then stirred for 15 minutes at room temperature beforeadding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 7.28 g, 38mmol) and n-decyll alcohol (4.88 mL, 4.04 g, 25.5 mmol). The cloudiywhite reaction mixture became a clear solution after stirring for 20minutes. Agitation was continued for 20 h at room temperature untilreaction was complete by HPLC analysis. DI water (100 mL) was charged toquench the reaction and the pH was adjusted to 6-7 using 1N HCl. Thelayers were separated, aqueous was extracted with dichloromethane (25mL), before combining the organic phases and drying over sodium sulfate.After filtration and concentration to dryness under vacuum a clearyellow oil was obtained which was purified by column chromatography (20to 60% ethyl acetate in heptane). A total of 11.2 g (22.42 mmol, 88.6%)was recovered as a thick yellow oil with 98.9 A % purity by HPLC.

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyric aiddodecyl ester (bendamustine C₁₂ ester)

Method A: A 250 mL three neck round bottom flask was equipped with anoverhead stirrer, thermocouple, temperature controller and nitrogensweep then charged with 10.0 g (25.34 mmol) of bendamustinehydrochloride, 4.77 g (25.6 mmol, 1.01 eq) of 1-dodecanol, 5.3 g (25.6mmol, 1.01 eq) of dicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31g (2.54 mmol, 0.1 eq) of DMAP. The reaction was stirred at roomtemperature overnight at which time an in process analysis indicated thereaction was complete. Solids were removed by vacuum filtration andwashed with 25 mL of MDC. The filtrate was washed with saturated aqueoussodium bicarbonate solution (2×100 mL), DI water (1×100 mL) and brine(1×100 mL) before drying over sodium sulfate, filtering andconcentrating to dryness in vacuo to an off-white semisolid. This solidwas triturated with 25 mL of MDC and the solid impurities were removedby vacuum filtration and washed with 5 mL of MDC. The filtrate wasconcentrated to dryness in vacuo to yield 11.53 g (21.9 mmol, 86.4%) ofthe product as an off-white semisolid with an HPLC purity of 93.7 A %.

Method B: A 20 liter jacketed cylindrical ChemGlass reaction vesselequipped with a thermocouple, heater/chiller, nitrogen inlet/outlet,addition funnel, and vacuum line was charged with the free base ofbendamustine (374 g, 1.04 mol, 1.0 eq.),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 300 g, 1.57 mol,1.5 equivalents), and dichloromethane (DCM, 3.74 L, 10 volumes). Whilestirring 1-dodecanol (292.1 g, 1.57 mol, 1.5 equivalents) was added. Thereaction mixture was heated to then stirred at 27° C. for 4 hours. Thebatch was cooled and held at 20° C. overnight. The batch was then washedwith 3.75 L of water and the layers were separated. The aqueous portionwas re-extracted with 1.2 L of dichloromethane, and the combineddichloromethane portions were dried over Na₂SO₄. After filtering toremove the drying agent, the filtrate was concentrated in vacuo toproduce the product as a crude oil. Another batch of this reaction with374 g of free base of bendamustine under the same conditions was carriedout. The crude products from the two batches were combined, mixed with4494 mL of heptanes and heated to 45-50° C. The resultant solution wasallowed to slowly cool to room temperature, precipitating an off-whitesolid. The slurry was stirred overnight at room temperature and thesolid was isolated at 10° C. by vacuum filtration. The wet cake waswashed with 1 L of heptanes and reslurried with 2.5 L of heptane at20-22° C. overnight. The product was collected by filtration and washedtwice with 500 mL of heptanes each time. Drying the wet cakes overnightat 20-22° C. yielded 653 g (59% yield) of white solids at 99.0 A % byHPLC. ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.8 Hz, 1H), 6.92 (d, J=2.3Hz, 1H), 6.78 (dd, J=8.8, 2.3 Hz, 1H), 3.99 (t, J=6.64 Hz, 2H), 3.70 (brs, 8H), 3.65 (s, 3H), 2.83 (t, J=7.4 Hz, 2H), 2.45 (t, J=7.4 Hz, 2H,overlapped partially with DMSO), 2.01 (quint, J=7.4 Hz, 2H), 1.54(quint, J=6.9 Hz, 2H), 1.24 (m, 18H), 0.85 (t, J=6.8 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid tetradecyl ester (bendamustine C₁₄ ester)

Method A: A 500 mL three necked round bottom flask equipped with a stirbar, thermocouple and nitrogen in/outlet was charged with 10.0 g (25.3mmol) of bendamustine hydrochloride, 6.5 g (30.4 mmol, 1.2 eq) ofteradecyl alcohol, 6.3 g (30.4 mmol, 1.2 eq) of dicyclohexylcarbodiimide (DCC), 100 mL of dichloromethane and 0.62 g (5.1 mmol, 0.2eq) of N,N-dimethylamino pyridine (DMAP). The reaction mixture wasstirred at room temperature for 20 hours at which time an HPLC analysisindicated the reaction was complete. Solids were removed by vacuumfiltration and the wetcake was washed with 50 mL of dichloromethanebefore concentrating the filtrate to dryness in vacuo. The resultantlight orange oil was triturated with 50 mL of dichloromethane and theundesired solids were removed by vacuum filtration. The filtrate wasonce again concentrated to dryness in vacuo to yield 16.5 g of asemisolid which was shown by ¹H NMR to contain tetradecanol and DMAP.The residue was chromatographed on 150 g of silica gel 60, 230-400 mesheluting with 1500 mL of MDC, 1000 mL of 0.5% methanol/MDC and 2000 mL of1% methanol/MDC collecting 100-150 mL fractions. Fractions containingthe desired product were combined and concentrated to dryness in vacuo.The residue was again triturated with 30 mL of MDC and the undesiredsolids removed by filtration. The filtrate was concentrated in vacuo toyield 5.0 g (9.2 mmol, 36.4%) of the desired product as a light purplesolid with a purity of 95.0 A %.

Method B: To a 150 mL three-neck glass vessel equipped with athermocouple, condenser, nitrogen inlet/outlet, and overhead mechanicalstirrer was charged the free base of bendamustine (16.0 g, 44.6 mmol,1.0 eq.), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 9.42 g,49.2 mol), 1-tetradecanol (10.6 g, 49.2 mmol) and dichloromethane (120mL). The reaction mixture was stirred at 27° C. overnight. The reactionsolution was cooled to room temperature and washed with 100 mL of water.While stirring, 1M aqueous HCl solution was added to adjust the pH ofthe aqueous layer to pH 3-4. The layers were separated. The aqueousportion was re-extracted with 100 mL of dichloromethane, and thecombined dichloromethane portions were dried over MgSO₄. After filteringto remove the drying agent, the filtrate was concentrated in vacuo toproduce the product as a waxy yellow solid. The solid was slurried in 80mL of heptanes at room temperature overnight. The product was collectedby filtration and dried, giving a white solid, 20.6 g (83.4% yield) with97.2 A % purity by HPLC. ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.8 Hz,1H), 6.91 (d, J=2.3 Hz, 1H), 6.78 (dd, J=8.8, 2.3 Hz, 1H), 3.98 (t,J=6.64 Hz, 2H), 3.70 (br s, 8H), 3.66 (s, 3H), 2.83 (t, J=7.4 Hz, 2H),2.45 (t, J=7.4 Hz, 2H, overlapped partially with DMSO), 2.01 (quint,J=7.3 Hz, 2H), 1.54 (m, 2H), 1.23 (m, 18H), 0.85 (t, J=7.12 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid pentadecyl ester (bendamustine C₁₅ ester)

A 250 mL three neck round bottom flask was equipped with an overheadstirrer, thermocouple, temperature controller and nitrogen sweep thencharged with 10.0 g (25.34 mmol) of bendamustine hydrochloride, 5.85 g(25.6 mmol, 1.01 eq) of pentadecanol, 5.3 g (25.6 mmol, 1.01 eq) ofdicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31 g (2.54 mmol, 0.1eq) of DMAP. The reaction was stirred at room temperature overnight atwhich time an in process analysis indicated the reaction was complete.Solids were removed by vacuum filtration and washed with 25 mL of MDC.The filtrate was washed with saturated aqueous sodium bicarbonatesolution (2×100 mL), DI water (1×100 mL) and brine (1×100 mL) beforedrying over sodium sulfate, filtering and concentrating to dryness invacuo to an off-white solid. This solid was triturated with 25 mL of MDCand the solid impurities were removed by vacuum filtration and washedwith 5 mL of MDC. The filtrate was concentrated to dryness in vacuo toyield 10.8 g (19.0 mmol, 75%) of the product as an off-white solid withan HPLC purity of 94.6 A %. ¹H NMR (400 MHz, CDCl₃) δ 7.17 (d, J=8.76Hz, 1H), 7.08 (d, J=2.32 Hz, 1H), 6.78 (dd, J=2.4, 8.76 Hz, 1H), 4.05(t, J=6.8 Hz, 2H), 3.72 (m, 4H), 3.69 (s, 3H), 3.63 (m, 4H), 2.91 (t,J=7.4 Hz, 2H), 2.49 (t, J=7.08 Hz, 2H), 2.18 (m, 2H), 1.60 (m, 2H), 1.32(m, 24H), 0.88 (t, J=6.68 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid hexadecyl ester (bendamustine C₁₆ ester)

A 250 mL three neck round bottom flask was equipped with an overheadstirrer, thermocouple, temperature controller and nitrogen sweep thencharged with 10.0 g (25.34 mmol) of bendamustine hydrochloride, 6.2 g(25.6 mmol, 1.01 eq) of hexadecanol, 5.3 g (25.6 mmol, 1.01 eq) ofdicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31 g (2.54 mmol, 0.1eq) of DMAP. The reaction was stirred at room temperature overnight atwhich time an in process analysis indicated the reaction was complete.Solids were removed by vacuum filtration and washed with 25 mL of MDC.The filtrate was washed with saturated aqueous sodium bicarbonatesolution (2×100 mL), DI water (1×100 mL) and brine (1×100 mL) beforedrying over sodium sulfate, filtering and concentrating to dryness invacuo to an off-white solid. This solid was triturated with 25 mL of MDCand the solid impurities were removed by vacuum filtration and washedwith 5 mL of MDC. The filtrate was concentrated to dryness in vacuo toyield 13.1 g (22.5 mmol, 88.8%) of the product as an off-white solidwith an HPLC purity of 94.0 A %. ¹H NMR (400 MHz, CDCl₃) δ 7.17 (d,J=8.76 Hz, 1H), 7.08 (d, J=2.32 Hz, 1H), 6.78 (dd, J=2.36, 8.72 Hz, 1H),4.05 (t, J=6.8 Hz, 2H), 3.72 (m, 4H), 3.69 (s, 3H), 3.63 (m, 4H), 2.91(t, J=7.4 Hz, 2H), 2.49 (t, J=7.08 Hz, 2H), 2.18 (m, 2H), 1.60 (m, 2H),1.32 (m, 26H), 0.88 (t, J=6.68 Hz, 3H).

Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid Oleoyl ester (bendamustine C₁₈ ester)

Method A: To a 250 mL, three neck, round bottom flask equipped with anoverhead stirrer, condenser with nitrogen sweep, heating mantle withtemperature controller and thermocouple was charged 1-octadecanol (50 g,185 mmol, 7.3 eq). The solid was heated to melt it before adding slowly4-(5-amino-1-methyl-1H-benzoimidazol-2-yl)-butyric acid (10 g, 25.3 mmol1.0 eq) and sulfuric acid (0.5 mL). The resulting slurry was stirred at70° C. for 6 hours and then cooled to 56° C., where methylene chloride(150 mL) was added. The reaction mixture was cooled to room temperatureand washed with water (100 mL). After phase separation, anotherextraction was performed with methylene chloride (100 mL). The organicphases were combined and dried over MgSO₄. The drying agent was removedby filtration. The filtrate was concentrated and subjected to SFCisolation. A white solid was obtained from evaporation of solvent in theSFC fractions under reduced pressure and dried with house vacuum at roomtemperature for 5 days, giving 1.2 g of the desired product in 7.1%yield and with 95.4 A % purity. ¹H NMR (400 MHz, CDCl3) δ 7.18 (d, J=8.8Hz, 1H), 7.09 (d, J=2.3 Hz, 1H), 6.78 (dd, J=8.8, 2.4 Hz, 1H), 4.05 (t,J=6.8 Hz, 2H), 3.74 (m, 7H), 3.62 (m, 4H), 2.93 (t, J=7.4 Hz, 2H), 2.49(t, J=7.1 Hz, 2H), 2.22 (quint, J=7.1 Hz, 2H), 1.60 (quint, J=7.1 Hz,2H), 1.28 (m, 30H), 0.88 (t, J=7.1 Hz, 3H); LC/MS (ESI, m/z) 610 (M+1).

Method B: To a 500 mL, three neck, round bottom flask equipped with astir bar, nitrogen sweep, and thermocouple was charged with bendamustineHCl (5.04 g, 12.8 mmol), 1-octadecanol (4.15 g, 15.3 mmol),N,N′-Dicyclohexylcarbodiimide (DCC, 3.17 g, 15.4 mmol),4-Dimethylaminopyridine (DMAP, 0.31 g, 2.56 mmol) and methylene chloride(250 mL). The resulting slurry was stirred at room temperature for 16hours. A solid was produced and removed from the reaction mixture byfiltration. The filtrate was washed with water (150 mL). After phaseseparation, the organic phase dried over MgSO₄. The drying agent wasremoved by filtration and the filtrate was concentrated and subjected toISCO chromatographic purification with a mixture of EtOAc and heptanes,giving a white solid 5.68 g (73% yield) with 99 A % purity.

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid docosyl ester (bendamustine C₂₂ ester)

A 250 mL three necked round bottom flask equipped with a stir bar,thermocouple and nitrogen in/outlet was charged with 5.0 g (12.7 mmol)of bendamustine hydrochloride, 4.2 g (12.9 mmol, 1.01 eq) of teradecylalcohol, 2.7 g (12.9 mmol, 1.01 eq) of dicyclohexyl carbodiimide (DCC),50 mL of dichloromethane (MDC) and 0.16 g (1.27 mmol, 0.1 eq) ofN,N-dimethylamino pyridine (DMAP). The reaction mixture was stirred atroom temperature overnight at which time an HPLC analysis indicated thereaction was complete. Solids were removed by vacuum filtration and thewetcake was washed with 50 mL. Two alternate purification procedureswere developed. The filtrate was washed with DI water (2×150 mL), diredover sodium sulfate, filtered and concentrated to dryness in vacuo. Theresultant waxy residue was chromatographed on 80 g of silica gel 60,230-400 mesh eluting with a gradient beginning with 100% MDC, then 0.5%methanol/MDC and finally 1% methanol/MDC collecting 100-150 mLfractions. Fractions containing the desired product were combined andconcentrated to dryness in vacuo. The residue was again triturated with20 mL of MDC and the undesired solids removed by filtration. Thefiltrate was concentrated in vacuo to yield 3.65 g (5.5 mmol, 43.1%) ofthe desired product as a waxy white solid with a purity of 95.7 A %. ¹HNMR (400 MHz, CDCl₃) δ 7.17 (d, J=8.72 Hz, 1H), 7.08 (d, J=2.28 Hz, 1H),6.78 (dd, J=2.36, 8.72 Hz, 1H), 4.05 (t, J=6.76 Hz, 2H), 3.72 (m, 4H),3.70 (s, 3H), 3.63 (m, 4H), 2.91 (t, J=7.44 Hz, 2H), 2.49 (t, J=7.08 Hz,2H), 2.18 (m, 2H), 1.60 (m, 2H), 1.32 (m, 38H), 0.88 (t, J=6.64 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid 2-dodecyl ester (branched bendamustine C₁₂ ester)

A 250 mL three neck round bottom flask was equipped with an overheadstirrer, thermocouple, temperature controller and nitrogen sweep thencharged with 10.0 g (25.34 mmol) of bendamustine hydrochloride, 4.77 g(5.75 mL, 25.6 mmol, 1.01 eq) of 2-dodecanol, 5.3 g (25.6 mmol, 1.01 eq)of dicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31 g (2.54 mmol,0.1 eq) of DMAP. The reaction was stirred at room temperature overnightat which time an in process analysis indicated the reaction wascomplete. Solids were removed by vacuum filtration and washed with 25 mLof MDC. The filtrate was diluted with 200 mL of MDC then washed with 4%aqueous sodium bicarbonate solution (1×500 mL) before drying over sodiumsulfate, filtering and concentrating to dryness in vacuo to an off-whitesolid. This solid was triturated with 25 mL of MDC and the solidimpurities were removed by vacuum filtration and washed with 5 mL ofMDC. The filtrate was concentrated to dryness in vacuo to yield thecrude product which was shown to contain residual 2-dodecanol by ¹H NMR.The crude product was chromatographed using 100 g of silica gel 60,230-400 mesh, eluting with first 1 L of heptanes, then 500 mL of 3:1heptane/EtOAc, 500 of 2:1 heptane/EtOAc and finally 500 mL of 1:1heptane/EtOAc collecting 100 mL fractions. Product containing fractionswere combined and concentrated to dryness in vacuo to yield 5.35 g(10.16 mmol, 40%) of the product as a light purple viscous oil with anHPLC purity of 99.5 A %. ¹H NMR (400 MHz, DMSO-d₆) δ 7.31 (d, J=8.76 Hz,1H), 6.93 (d, J=2.28 Hz, 1H), 6.78 (dd, J=2.36, 8.76 Hz, 1H), 4.8 (m,1H), 3.7 (s, 8H), 3.65 (s, 3H), 2.82 (t, J=7.4 Hz, 2H), 2.42 (t, J=7.36Hz, 2H), 2.00 (m, 2H), 1.50 (m, 2H), 1.25 (s, b, 16H), 1.14 (d, J=6.24,2H), 0.84 (t, J=6.68 Hz, 3H).

Preparation of Bendamustine C₁₂ Ester

A 20 liter jacketed cylindrical ChemGlass reaction vessel equipped withthermocouple, heater/chiller, nitrogen inlet, addition funnel,condenser, and vacuum line was charged with a slurry of 428 g (1.10mmol) of pretreated bendamustine hydrochloride in 10 volumes of trace GCanalysis grade methylene chloride. Agitation was set at 100 RPM and thejacket was set at 20° C. To this mixture was added diisopropylethylamine(213 ml, 1.1 eq) via an addition funnel over 10 minutes. After a 34minute hold, melted dodecanol (227 g, 1.1 eq) was added in one portion.After an 11 minute hold, EDCI (320.3 g, 1.5 eq) was added to the batch.The resulting clear yellow solution was agitated for 23.5 hours at ˜20°C. At this point, an IPC indicated 0.54% starting starting materialremained. Ten volumes of water were added and the reaction was agitatedfor an additional 15 minutes. The lower organic layer was drained,filtered through a 5 micron filter cartridge, and the filter cartridgewas rinsed with 1 volume of GC analysis grade methylene chloride. Themethylene chloride solution was concentrated in vacuo to afford thecrude product as a viscous yellow oil. Five volumes of filteredn-heptane were added to the oil and the mixture was concentrated invacuo to remove residual methylene chloride to yield 615 g of crudesolids with in 92.9 wt % translating to a 98.0% yield. Final purity was98.4% on a dry basis.

Purification of Bendamustine C₁₂ Ester

Crude CEP-40125 (1100 g API, 1250 g crude) was taken up in n-heptane (6volumes) and transferred to a 20 liter jacketed cylindrical ChemGlassreaction vessel equipped with thermocouple, heater/chiller, nitrogeninlet, condenser, and vacuum line. The slurry was warmed to 40° C. todissolve all solids. Upon reaching 32.6° C., dissolution occurred. Thereaction mixture was then cooled to 17.7° C. over 2.5 hours, at whichpoint the product precipitated. The reaction mixture was then re-warmedto 23° C. to dissolve fine particles over 26 minutes, and cooled to 4°C. over 2 hours. The solids were filtered through a sealed filter andwashed with 2 volumes of cold n-heptane over 4.5 hours. An IPC indicated0.20% residual dodecanol. The solids were dried under vacuum for 24hours at 30° C. to constant weight to afford 1018 g CEP-40125 in 98.3%purity, representing a 92.5% yield.

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid 5-decyl ester (branched bendamustine C₁₀ ester)

A 250 mL three neck round bottom flask was equipped with an overheadstirrer, thermocouple, temperature controller and nitrogen sweep thencharged with 3.0 g (7.6 mmol) of bendamustine hydrochloride, 1.21 g (7.7mmol, 1.01 eq) of 5-decanol, 1.59 g (7.7 mmol, 1.01 eq) ofdicyclohexylcarbodiimide (DCC), 30 mL of 1,2-ethylene dichloride (EDC)and 0.1 g (0.76 mmol, 0.1 eq) of DMAP. The reaction was stirred at 75°C. for five days until an in process analysis indicated the reaction wascomplete. Solids were removed by vacuum filtration and washed with 5 mLof EDC. The filtrate was washed with 4% aqueous sodium bicarbonatesolution (1×50 mL) before drying over sodium sulfate, filtering andconcentrating to dryness in vacuo. The residue was combined with theresidue from a 10 g batch run carried out under the same conditions andthe combined batches were chromatographed. The chromatography wascarried out using 100 g of silica gel 60, 230-400 mesh, eluting withfirst 2 L of heptanes, then 1 L of 3:1 heptane/EtOAc and 1 L of 2:1heptane/EtOAc collecting 100 mL fractions. Product containing fractionswere combined and concentrated to dryness in vacuo to yield 3.97 g (7.96mmol, 24.2%) of the product as a clear yellow oil with an HPLC purity of99.4 A %. ¹H NMR (400 MHz, DMSO-d₆) δ 7.31 (d, J=8.76 Hz, 1H), 6.93 (d,J=2.28 Hz, 1H), 6.78 (dd, J=2.40, 8.80 Hz, 1H), 4.8 (m, 1H), 3.7 (s,8H), 3.65 (s, 3H), 2.83 (t, J=7.4 Hz, 2H), 2.44 (t, J=7.36 Hz, 2H), 2.02(m, 2H), 1.48 (m, 4H), 1.25 (s, b, 10H), 0.84 (m, 6H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid cyclohexyl ester

A 250 mL three neck round bottom flask was equipped with an overheadstirrer, thermocouple, temperature controller and nitrogen sweep thencharged with 10 g (25.34 mmol) of bendamustine hydrochloride, 2.56 g(2.7 mL, 25.6 mmol, 1.01 eq) of cyclohexanol, 5.3 g (25.6 mmol, 1.01 eq)of dicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31 g (2.54 mmol,0.1 eq) of DMAP. The reaction slurry was stirred at RT for 18 h until anin process analysis indicated the reaction was complete. Two new majorproduct peaks were observed. Solids were removed by vacuum filtrationand washed with 5 mL of MDC. The filtrate was washed with 4% aqueoussodium bicarbonate solution (2×100 mL) before drying over sodiumsulfate, filtering and concentrating to dryness in vacuo to yield ayellow oil with some solids present. ¹H NMR analysis indicated residualDMAP and cyclohexanol was along with DCC by-products were present inaddition to the desired product. The residue was slurried with 50 mL ofMDC to remove residual cylohexanol then concentrated in vacuo andchromatographed. The chromatography was carried out using 50 g of silicagel 60, 230-400 mesh, eluting with 1:1 heptane/EtOAc collecting 100 mLfractions. Product containing fractions were combined and concentratedto dryness in vacuo to yield 3.11 g (7.06 mmol, 27.9%) of the product asan off-white solid with an HPLC purity of 97.8 A %. ¹H NMR (400 MHz,DMSO-d₆) δ 7.31 (d, J=8.76 Hz, 1H), 6.93 (d, J=2.28 Hz, 1H), 6.77 (dd,J=2.40, 8.80 Hz, 1H), 4.65 (m, 1H), 3.7 (s, 8H), 3.65 (s, 3H), 2.83 (t,J=7.4 Hz, 2H), 2.44 (t, J=7.36 Hz, 2H), 2.00 (m, 2H), 1.76 (m, 4H), 1.65(m, 2H), 1.33 (m, b, 6H).

Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid PEG-2000 ester (bendamustine PEG-2000 ester)

To a 100 mL three-neck glass vessel equipped with a stir bar,thermocouple, condenser, and nitrogen inlet/outlet was chargedbendamustine hydrochloride (2.0 g, 5.1 mmol, 1.0 eq.) anddichloromethane (30 mL). Triethyl amine (0.71 mL, 5.1 mmol) was added tothe slurry at 22° C. and stirred for 20 minutes. Methoxypolyethyleneglycol 2000 (PEG-OMe-2000, 12.2 g, 6.1 mmol) and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 1.5 g, 7.6 mol)were added. The reaction mixture was stirred at 22° C. for 5.5 hours, atthis point addition of PEG-OMe-2000 (1.0 g) was followed by stirring for3 days through weekend. Water (20 mL) was added and pH was adjusted topH 5-6 by adding 1M hydrochloric acid. The phases separated slowly. Theaqueous portion was re-extracted with 20 mL of dichloromethane, and thecombined dichloromethane portions were dried over MgSO₄. After filteringto remove the drying reagent, the filtrate was concentrated in vacuo toproduce the product as a waxy solid. The solid was slurried in 10 mL ofheptanes at room temperature. The product was collected by filtrationand dried at 30° C. under vacuum, giving a white and powdery solid, 11.7g (99% yield) with 97.9 A % purity by HPLC. ¹H NMR (400 MHz, DMSO-d6) δ7.32 (d, J=8.6 Hz, 1H), 6.92 (d, J=2.2 Hz, 1H), 6.78 (dd, J=8.8, 2.2 Hz,1H), 4.12 (t, J=4.8 Hz, 2H), 3.70 (m, 12H), 3.60 (m, 3H), 3.51 (m,224H), 3.43 (m, 4H), 3.32 (m, 58H), 2.84 (t, J=7.4 Hz, 2H), 2.45 (2H,overlapped partially with DMSO), 2.01 (quint, J=7.3 Hz, 2H).

Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid PEG-5000 ester (bendamustine PEG-5000 ester)

To a 50 mL three-neck glass vessel equipped with a stir bar,thermocouple, condenser, and nitrogen inlet/outlet was chargedbendamustine hydrochloride (0.5 g, 1.27 mmol, 1.0 eq.) anddichloromethane (15 mL). Triethyl amine (0.18 mL, 1.28 mmol) was addedto the slurry at 22° C. and stirred for 20 minutes along withmethoxypolyethylene glycol 5000 (PEG-OMe-5000, 6.33 g, 1.27 mmol) and,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 0.36 g, 1.88 mol).The reaction mixture was stirred at 22° C. overnight. Water (20 mL) wasadded and pH was adjusted to pH 3-4 by adding 1M hydrochloric acid. Thephases separated slowly. The aqueous portion was re-extracted with 10 mLof dichloromethane. The combined dichloromethane portions were washedwith brine (20 mL) and dried over MgSO₄. After filtering to remove thedrying agent, the filtrate was concentrated in vacuo to produce theproduct as a waxy solid. The solid was slurried in 20 mL of heptanes atroom temperature. The product was collected by filtration and dried at30° C. under vacuum, giving a white and powdery solid, 5.24 g (77%yield) with 99 A % purity by HPLC. ¹H NMR (400 MHz, DMSO-d6) δ 7.38 (d,J=12 Hz, 1H), 6.91 (d, J=2.4 Hz, 1H), 6.82 (d, J=10 Hz, 1H), 4.12 (t,J=4.7 Hz, 2H), 3.72 (br s, 8H), 3.68 (m, 5H), 3.59 (m, 3H), 3.51 (m,436H), 3.42 (m, 3H), 3.31 (m, 28H), 2.88 (t, J=7.4 Hz, 2H), 2.45 (t, 2H,overlapped partially with DMSO), 2.01 (quint, J=7.3 Hz, 2H).

General Procedure for Transesterification of Bendamustine Methyl Ester

To a 50 mL three-neck glass vessel equipped with a stir bar,thermocouple, condenser with a Dean-Stark trap, and nitrogeninlet/outlet was charged bendamustine methyl ester (0.5 g, 1.27 mmol,1.0 eq.), catalyst (0.05-1.4 eq.), an appropriate solvent (5-15volumes), and an excess of dodecanol (5-10 eq.). The resulting reactionmixture was heated to reflux and monitored by HPLC. The results undervaried conditions are summarized below.

Dean- Start Catalyst Solvent Alcohol Temp Stark Time material ProductCatalyst (eq) Solvent Volumes Equivs (° C.) Trap (h) (HPLC A %) (HPLC A%) CH₃SO₃H 1.40 NA NA 2.50 30 N 3 3.8 94.5 CH₃SO₃H 1.40 DCM 10 2.50 30 N47 22.0 76.2 CH₃SO₃H 1.40 Toluene 10 2.50 70 N 24 19.3 69.7 I₂ 0.25Toluene 5 2.50 75 N 7 96.0 0.2 TiO(acac)₂ 0.05 Xylenes 10 1.01 130 N 792.7 4.0 TiO(acac)₂ 0.10 Xylenes 5 10.0 130 N 6 10.5 85.5 TiO(acac)₂0.10 Xylenes 5 10.0 160 N 5 9.0 78.6 TiO(acac)₂ 0.10 Xylenes 15 5.00 160Y 6 1.0 97.0 H₂SO₄ 0.5 Toluene 10 5.0 100 N 23 78.9 13.5 DMAP 0.5Toluene 10 5.0 100 N 23 83 0 p-TSA 0.5 Xylenes 10 5.0 130 N 21 67.2 15.6Sm(Oi-Pr)₃ 0.5 THF 10 5.0 25-45 N 27 2.6 90.5 Superbase 0.5 THF 10 5.025-45 N 27 3.3 49.8 Sm(Oi-Pr)₃ 0.1 Xylenes 15 5.0 160 N 8 72.8 24.3Sm(Oi-Pr)₃ 0.5 THF 10 5.0 40 N 16 95.8 2.5 TiO(acac)₂ 0.05 Xylenes 155.0 150 Y 3 0.3 96.9 TiO(acac)₂ 0.05 Toluene 15 5.0 105 N 70 78.9 15.9TiO(acac)₂ 0.10 Toluene 15 5.0 115 Y 22 ND 96.4 TiO(acac)₂ 0.05 Toluene15 5.0 130 N 24 83.2 4.1 TiO(acac)₂ 0.05 Toluene 15 5.0 115 Y 21 0.497.7

Preparation of Bendamustine Hydrochloride Amides4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-N-decyl-butryamide(bendamustine C₁₀ amide)

A 250 mL three neck round bottom flask equipped with a stir bar,thermocouple, cooling bath, 60 mL pressure equalizing dropping funneland nitrogen in/outlet was charged with 10.0 g (25.3 mmol) ofbendamustine hydrochloride, 10.6 g (27.8 mmol) of HATU and 100 mL ofN,N-dimethylfomramide (DMF). To this stirred yellow solution was added4.41 mL (3.27 g, 25.3 mmol) of N,N-diisopropylethyelamine (DIPEA). Anexotherm to 27.1° C. was noted and the solution became a darker yellow.The reaction was cooled to 6.6° C. where a solution of 6.2 mL (4.59 g,35.5 mmol) of DIPEA, 5.11 mL (4.1 g, 25.6 mmol) of decyl amine in 20 mLof DMF was added drop-wise over 13 min at 2.7-7.6° C. Once addition wascomplete the reaction was allowed to stir at <10° C. for 1.5 hours atwhich time an in process analysis indicated the reaction was complete.The batch was quenched onto 200 mL of DI water and extracted with ethylacetate (2×175 mL). The organic phases were combined, washed with 10%sodium hydrogen phosphate (1×200 mL), 8% aqueous sodium bicarbonate(1×200 mL) and brine (1×200 mL) before concentrating to dryness in vacuoto give a sticky white solid. This solid was triturated with heptanes(75 mL) and became a flowable solid which was isolated by vacuumfiltration. The wetcake was dried in a vacuum oven at 25° C. overnightto yield 13.33 g (25.3 mmol, 100%) of the desired product as a whitesolid with an HPLC purity of 98.01 A %. ¹H NMR (400 MHz, DMSO-d6) δ 7.72(s, b, 1H), 7.33 (d, J=8.76 Hz, 1H), 6.91 (d, J=2.28 Hz, 1H), 6.80 (dd,J=2.36, 8.8 Hz), 3.7 (s, 8H), 3.66 (s, 3H), 3.01 (q, J=6.8, 12.68, 2H),2.79 (t, J=7.44 Hz, 2H), 2.18 (t, J=7.36 Hz, 2H), 1.95 (m, 2H), 1.36 (m,2H), 1.22 (s, b, 14), 0.84 (t, J=6.68 Hz, 3H).

4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-N-tetradecyl-butryamide(bendamustine C₁₄ amide)

A 250 mL three neck round bottom flask equipped with a stir bar,thermocouple, cooling bath, 60 mL pressure equalizing dropping funneland nitrogen in/outlet was charged with 10.0 g (25.3 mmol) ofbendamustine hydrochloride, 10.6 g (27.8 mmol) of HATU and 100 mL ofN,N-dimethylfomramide (DMF). To this stirred yellow solution was added4.41 mL (3.27 g, 25.3 mmol) of N,N-diisopropylethyelamine (DIPEA). Anexotherm to 27.1° C. was noted and the solution became a darker yellow.The reaction was cooled to 3.3° C. where a solution of 6.2 mL (4.59 g,35.5 mmol) of DIPEA, 5.75 gf (25.6 mmol) of tetradecyl amine in 40 mL ofDMF was added drop-wise over 6 min at <10° C. Once addition was completethe reaction became very thick and difficult to stir. It was transferredto a 500 mL three neck round bottom flask equipped with an overheadstirrer and thermocouple, then stirred at RT for three hours at whichtime an in process analysis indicated the reaction was complete. Thebatch was quenched onto 400 mL of DI water and extracted with ethylacetate (2×300 mL). the organic phases were combined, washed with 10%sodium hydrogen phosphate (1×300 mL), 8% aqueous sodium bicarbonate(1×300 mL) and brine (1×300 mL) before drying over sodium sulfate,filtering and concentrating to dryness in vacuo. The residue waspurified by chromatography using 100 g of silica gel 60, 230-400 mesh,eluting with 1% MeOH/MDC (2 L), 2.5% MeOH/MDC (1 L) and 5% MeOH/MDC (1L) collecting ˜100 mL fractions. The product containing fractions werecombined and concentrated to dryness in vacuo to yield 7.86 g (14.6mmol, 57.6%) of the desired product as a white solid with an HPLC purityof 97.3 A %. ¹H NMR (400 MHz, DMSO-d6) δ 7.72 (s, b, 1H), 7.33 (d,J=8.84 Hz, 1H), 6.91 (d, J=2.22 Hz, 1H), 6.80 (dd, J=2.36, 8.84 Hz),3.71 (s, 8H), 3.70 (s, 3H), 3.01 (q, J=6.8, 12.68, 2H), 2.79 (t, J=7.44Hz, 2H), 2.18 (t, J=7.36 Hz, 2H), 1.97 (m, 2H), 1.36 (m, 2H), 1.28 (s,b, 22), 0.84 (t, J=7.04 Hz, 3H).

4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-N-octadecyl-butyramide(bendamustine C₁₈ amide)

A 250 mL three neck round bottom flask equipped with a stir bar,thermocouple, cooling bath, 60 mL pressure equalizing dropping funneland nitrogen in/outlet was charged with 10.0 g (25.3 mmol) ofbendamustine hydrochloride, 10.6 g (27.8 mmol) of HATU and 100 mL ofN,N-dimethylfomramide (DMF). To this stirred yellow solution was added4.41 mL (3.27 g, 25.3 mmol) of N,N-diisopropylethyelamine (DIPEA). Anexotherm to 27.1° C. was noted and the solution became a darker yellow.The reaction was cooled to 2.0° C. where a suspension of 6.2 mL (4.59 g,35.5 mmol) of DIPEA, 7.65 g (25.6 mmol) of octadecyl amine in 0 mL ofDMF was added via pipet. Once addition was complete the reaction becamevery thick and difficult to stir. It was warmed to room temperature andthe magnetic stir bar was replaced with an overhead stirrer. The batchwas stirred at RT overnight after which time an in process analysisindicated the reaction was complete. The batch was quenched onto 300 mLof DI water and extracted with dichloromethane (2×150 mL). The organicphases were combined, washed with 10% sodium hydrogen phosphate (1×300mL), 8% aqueous sodium bicarbonate (1×300 mL) and brine (1×300 mL)before drying over sodium sulfate, filtering and concentrating todryness in vacuo. The residue was purified by chromatography using 100 gof silica gel 60, 230-400 mesh, eluting with 1% MeOH/MDC (2 L), 2.5%MeOH/MDC (1 L) and 5% MeOH/MDC (1 L) collecting ˜100 mL fractions. Theproduct containing fractions were combined and concentrated to drynessin vacuo to yield 5.11 g (8.38 mmol, 33%) of the desired product as awhite solid with an HPLC purity of 90.9 A %. The major impurity wasshown to be the C-16 amide which results from an impurity in thestarting amine ¹H NMR (400 MHz, DMSO-d6) δ 7.72 (s, b, 1H), 7.33 (d,J=8.84 Hz, 1H), 6.91 (d, J=2.22 Hz, 1H), 6.80 (dd, J=2.36, 8.84 Hz),3.71 (s, 8H), 3.70 (s, 3H), 3.01 (q, J=6.8, 12.68, 2H), 2.79 (t, J=7.44Hz, 2H), 2.18 (t, J=7.36 Hz, 2H), 1.97 (m, 2H), 1.36 (m, 2H), 1.28 (s,b, 30H), 0.85 (t, J=6.32 Hz, 3H).

(S)-2-(4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyrylamino)-propionicacid methyl ester

A 250 mL three neck round bottom flask equipped with a stir bar,thermocouple, cooling bath, addition funnel and nitrogen in/outlet wascharged with 10 g (25.3 mmol) of bendamustine hydrochloride, 10.6 g(27.8 mmol) of HATU and 100 mL of DMF. The batch was cooled to 1.9° C.where 8.8 mL (6.54 g, 50.6 mmol) of DIPEA was added over 2 minutes. Thereaction exothermed to 9° C. and became orange. A solution of 3.57 g(25.6 mmol) of L-alanine methyl ester hydrochloride and 6.2 mL (4.57 g,35.4 mmol) of DIPEA in 20 mL of DMF was added drop-wise over 10 minutesat 4.9-5.7° C. The reaction was slowly warmed to RT over one hour andstirred for three hours at which time an in process assay indicated thereaction was complete. The batch was quenched onto 400 mL of 1:1 ethylacetate/DI water. The layers were separated, the organic was washed with10% sodium hydrogen phosphate (1×200 mL), 8% sodium bicarbonate 91×200mL) and brine (1×200 mL), before drying over sodium sulfate, filteringand evaporating to dryness in vacuo. The residue was purified bychromatography using 100 g of silica gel 60, 230-400 mesh, eluting with1% MeOH/MDC (3 L), 2.5% MeOH/MDC (1 L) and 5% MeOH/MDC (500 mL)collecting ˜100 mL fractions. The product containing fractions werecombined and concentrated to dryness in vacuo to yield 7.1 g (16.0 mmol,63.3%) of the desired product as a white solid with an HPLC purity of97.4 A %. ¹H NMR (400 MHz, DMSO-d6) δ 8.25 (d, J=6.92 Hz), 1H), 7.40 (d,J=8.84 Hz, 1H), 6.92 (d, J=2.2 Hz, 1H), 6.86 (dd, J=2.32, 8.88 Hz), 4.25(q, 1H), 3.72 (s, 8H), 3.71 (s, 3H), 3.62 (s, 3H), 2.87 (t, J=7.48 Hz,2H), 2.25 (t, J=7.52 Hz, 2H), 1.99 (m, 2H), 1.26 (d, J=7.32 Hz, 3H).

General Procedure for Preparing Solution Formulations of BendamustineEsters of the Invention:

A stock solution of the bendamustine ester of the invention wasdissolved in a 60/40 (v/v) mixture of dimethylacetamide (“DMA”) andSolutol® HS-15 at about 100 mg/mL concentration. The mixture was stirredat room temperature until dissolved. The resulting stock solution, whichwas stable for several months, was diluted with 0.9% saline to thedesired concentration and dosed within about 2 hours.

Bendamustine C₁₄ Ester Solution Formulation:

A stock solution was prepared by dissolving 320.1 mg of bendamustine C₁₄ester in 4 mL of a 60/40 (v/v) mixture of DMA and Solutol® HS-15. Themixture was stirred for about 2 hours until dissolved. Prior to dosing,the stock solution was diluted by removing 1.00 mL of the stock solutionand adding 16.20 mL saline and stirring for 5 minutes at roomtemperature. The resulting formulation was 3 mg-equ/mL bendamustine.

Pretreatment to Remove Residual Ethanol:

Bendamustine hydrochloride (277 g) was charged into a 5 L evaporationflask followed by 2 volumes of DI water and 5 volumes of acetone. Thesolvents were distilled under vacuum at a maximum temperature of 40° C.over 5.5 hours. An additional 5 volumes of acetone were added and theresulting slurry was rotated at atmospheric pressure on the rotaryevaporator at 35° C. for 15 minutes. The batch was then filtered througha sealed sintered glass funnel and the resulting white solids wererinsed with 2 volumes of acetone. The solids were transferred to adrying tray and dried at 40° C. to constant weight for 17 hours beforeisolating 266 g (96.0%) of product in 99.8 A % purity with KF of 0.26%.

General Procedure for Preparing Human Serum Albumin (“HSA”) NanoparticleFormulations of Bendamustine Esters of the Invention

Nanoparticles were formed from an O/W emulsion using dichloromethane andHSA as a surfactant. The oil phase was prepared by dissolving thedesired amount of bendamustine ester in dichloromethane at aconcentration of about 120 mg/mL. The water phase was prepared bydissolving 2-4× the amount of HSA (w/w base on the bendamustine ester)in 5-15× the volume of water (w/w based on dichloromethane). Typically,mannitol was added to the aqueous phase at 5-10% to make the solutionisotonic for injection and to provide a pharmaceutically appropriateproduct post lyophilization. The O/W emulsion was formed by emulsifyingthe oil phase and the water phase using and IKA hand-held homogenizer atmedium intensity for about 30 seconds. The nanoparticles were formed byprocessing the crude O/W emulsion through a Microfluidizer® highpressure homogenizer (5 passes at about 30,000 psi) to provide 50-100 nmsized particles, as measured using dynamic light scattering (MalvernZetasizer). The solvent was removed under vacuum and the resultingconcentrate was either lyophilized or stored frozen prior to dosing.These formulations exhibited good physical and chemical stability.

The lyophilized nanoparticles were reconstituted and analyzed usingcryo-Transmission Electron Microscopy (c-TEM). The majority of thenanoparticles were 20-40 nm solid spheres that were readily dispersed inwater. A minority of particles were in the 125 nm range. The particlesall had a smooth surface.

Bendamustine C₁₂ Ester HSA Nanoparticles:

An oil phase was prepared by dissolving 600 mg of bendamustine C₁₂ esterin 5 mL of dichloromethane. The oil phase was emulsified with an aqueousphase comprised of 60 mL deionized (“DI”) water, 2.4 g HSA (lyophilizedsolid from Sigma-Aldrich, St. Louis, Mo.) and 6.6 g mannitol using anIKA Ultra-Turrex hand held homogenizer to obtain a coarse emulsion. Thisemulsion was passed five times through a Microfluidics M-110P highpressure homogenizer at about 30,000 psi. The dichloromethane wasremoved from the resulting nanoparticle suspension using arotory-evaporator and the resulting aqueous suspension was diluted tobring the total volume to 100 mL with DI water. This suspension was thenportioned in 10 mL aliquots into 30 mL serum vials and lyophilized.

Bendamustine C₁₂ Ester HSA Nanoparticles with Poly-Lactic Glycolic Acid(PLGA):

An oil phase was prepared by dissolving 300 mg of bendamustine C₁₂ esterand 500 mg of PLGA (50/50 lactic to glycolic with a MW of 7,000-17,000;Aldrich Part#719897) in 2.5 mL of dichloromethane. The oil phase wasemulsified with an aqueous phase comprised of 30 mL DI water, 1.2 g HSA(lyophilized solid from Sigma-Aldrich) and 3.3 g mannitol using and IKAUltra-Turrex hand held homogenizer to obtain a coarse emulsion. Thisemulsion was passed five times through a Microfluidics M-110P highpressure homogenizer at about 30,000 psi. The dichloromethane wasremoved from the resulting nanoparticle using a roto-evaporator and theresulting aqueous suspension was diluted to bring the total volume to 50mL with DI water. The resulting nanoparticles had a particle size of90.5 nm (Z_(avg)) as measured by Malvern Zetasizer. This suspension wasthen portioned in 10 mL aliquots into 30 mL serum vials and lyophilized.

Preparation of PEGylated Nanoparticle Formulations of BendamustineEsters of the Invention:

A series of nanoparticle formulations were prepared with a PEG coating,which was reported in the literature (Alexis, F., MolecularPharmaceutics, 5, (2008), 505-515) to provide a “stealth” coating andaid in the particle ability to avoid the body's immune system. Theincorporation of PEG was done by using a PEG based surfactant (copolymerof PEG and poly lactic acid) instead of HSA or using bioconjugatechemistry to covalently attach PEG groups to the free NH₂ groups on thesurface of the HSA nanoparticles. Both systems showed increased plasmacirculation times in PK studies.

Bendamustine C₁₂ Ester HSA Nanoparticles with Polyethyleneglycol (PEG)Coating:

An oil phase was prepared by dissolving 600 mg of bendamustine C₁₂ esterin 5 mL of dichloromethane. The oil phase was emulsified with an aqueousphase comprised of 60 mL DI water, 2.4 g HSA (lyophilized solid fromSigma-Aldrich) and 6.6 g mannitol using and IKA Ultra-Turrex hand heldhomogenizer to obtain a coarse emulsion. This emulsion was passed fivetimes through a Microfluidics M-110P high pressure homogenizer at˜30,000 psi. The dichloromethane was removed from the resultingnanoparticle suspension using a roto-evaporator and the resultingaqueous suspension was diluted to bring the total volume to 50 mL withDI water. The suspension of nanoparticles were then diluted into 200 mLof a 100 mM pH 8.5 borate buffer and the particle size andzeta-potential of the resulting nanoparticles were measured using aMalvern Zetasizer. The nanoparticles had a particle size of 78.9 nm(Z_(avg)) and a surface charge of −13.0 mV. The suspension was stirredand 150 mg of methoxy-PEG_(5,000)-n-hydroxysuccinimide ester (LaysanPolymer) was added. The reaction was mixed for ˜90 minutes at roomtemperature and the particle size and zeta-potential were re-measured.The nanoparticles particle size was found to be 83.6 nm (Zavg) and thezeta-potential was −7.35 mV. The nanoparticles were buffer exchanged andconcentrated with a 6.6% (wt/wt) mannitol solution and a 50,000 MWCOdiafiltration cartridge. This suspension was then portioned in 10 mLaliquots into 30 mL serum vials and lyophilized.

Bendamustine C₁₂ Ester Nanoparticles with Poly-Lactic Glycolic Acid(PLGA) and polyoxyEthylene Lactic Acid Copolymer (PELA) Surfactant:

An oil phase was prepared by dissolving 300 mg of bendamustine C₁₂ esterand 500 mg of PLGA (50/50 lactic to glycolic with a MW of 7,000-17,000;Aldrich Part#719897) in 2.5 mL of dichloromethane. The oil phase wasemulsified with an aqueous phase comprised of 30 mL DI water, 0.6 g acopolymer of PEG-5,000-poly lactic acid 1,000 (copolymer prepared usingprocedure from A. Lucke, Biomaterials, 21, (2000), 2361-2370) and 3.3 gmannitol using and IKA Ultra-Turrex hand held homogenizer to obtain acoarse emulsion. This emulsion was processed for 5 minutes through aMicrofluidics M-110P high pressure homogenizer at 30,000 PSI. Thedichloromethane was removed from the resulting nanoparticle suspensionusing a roto-evaporator and the resulting aqueous suspension was dilutedto bring the total volume to 50 mL with DI water. The resultingnanoparticles had a particle size of 248.0 nm (Z_(avg)) as measured byMalvern Zetasizer. This suspension was then portioned in 10 mL aliquotsinto 30 mL serum vials and lyophilized.

Bendamustine C₁₂ Ester HSA Nanoparticles from Concentrate:

An oil phase was prepared by dissolving 4.8 g of Bendamustine C₁₂ esterin 13.4 g of dichloromethane. The oil phase was emulsified with anaqueous phase comprised of 111 g deionized (“DI”) water and 9 g HSAusing an IKA Ultra-Turrex hand held homogenizer to obtain a coarseemulsion. This emulsion was passed five times through a MicrofluidicsM-110P high pressure homogenizer at about 30,000 psi. The resultingnanoemulsion concentrate was stabilized by adding 12 g NaCl and mixinguntil dissolved. Stabilization can also be achieved using other methodsknown in the art, for example, other salts, controlled heating, and/orpH adjustments. Cross-linking with, for example, glutaraldehyde, canalso assist in preventing aggregation.

The dichloromethane was removed from the resulting nanoparticlesuspension using a rotory-evaporator. The resulting aqueous suspensionwas mixed with 12 g of sucrose and DI water was added to bring the totalweight to 480 g. This suspension was then portioned in 7.5 mL aliquotsinto 20 mL serum vials and lyophilized.

Solutions of the HSA nanoparticles exhibited a slow particle sizeincrease with time. Some solutions reached ˜200 nm in size within 12-24hours as measured by dynamic light scattering. The nature of thisincrease was investigated using c-TEM in order to determine if thenanoparticles were aggregating or if excess protein in the formulationwas adding on to the surface of the particle to increase the particlesize. A vial of lyophilized nanoparticles was reconstituted and imagedusing c-TEM (FIG. 21). The same vial of nanoparticles was allowed tostand at room temperature for 24 hours then re-imaged (FIG. 22). Theoriginal sample contained spherical particles with a size distributionbetween 25-60 nm. The aged sample exhibited an increase in sizedistribution to 35-130 nm with no sign of particle aggregation. Bothsamples contain a population of 1-3 nm particles which are consistentwith “free” protein. These results suggest that “free” HSA is adding tothe surface of the nanoparticles while in solution to cause a slowincrease in particle size. Several strategies may be employed tomitigate this process including change in pH, change in osmolarity,solvent addition and diafiltration to remove excess protein.

Protocol for Preparing Tumor Cell Isolates:

Charles River Labs athymic nude mice bearing MDA-MB-231 breast carcinomacell line subcutaneous xenografts (5×10⁶ cells in matrigel) weresacrificed to make a tumor isolate. Using sterile instruments andworking aseptically, the tumors were removed from the euthanized animal.The tumor was placed in a 50 mL sterile conical tube and 5 mL of trypsinwas added. The tumor was cut into small pieces and incubated at 37° C.for 30 minutes. After incubation, a cell strainer was placed on asecond, sterile, 50 mL conical tube and the contents of the first tubewere placed in the cell strainer. The tissue was forced through thefilter using the flat end of a syringe plunger. The cell strainer waswashed with 5 mLs of media containing FBS. The cells were spun down andthe supernatant was discarded. The cells were resuspended in 20 mLs ofcomplete media containing P/S and placed in a 75 cm flask. This data isset forth in Table 1.

General Procedure for MTS Cell Assay:

Human carcinoma cells (2,000 cells/well) were incubated with the desiredconcentration of the test molecule (0-200 μM) for 72 hours. The cellswere then incubated with MTS solution (Promega) for 1-2 hours, and thecompounds effects on cell proliferation was determined by measuring theabsorbance at 490 nm. Cell growth was expressed as a percent of theappropriate control (placebo). This data was used to calculate an IC₅₀for each compound. All data reported was the mean±SE of threeindependent experiments. This data is set forth in Table 1.

TABLE 1

                    MB-231 (HBC)                     H-460 (NSCLC) R₁IC₅₀ (μM) R² IC₅₀ (μM) R² —OH 13-16 0.88-0.95 4-26 0.83-0.95 —CH₃ 660.95 1.2 0.9 —C₄H₉ 13.3 0.91 15.73 0.9 —C₆H₁₃ 43.7 0.93 7.4 0.89 —C₈H₁₇53.1 0.86 49 0.89 —C₁₀H₂₁ 38.89 0.87 23.76 0.93 —C₁₂H₂₅ 33.3 0.78 28.80.69 —C₁₄H₂₉ >200 0.83 129.9 0.87 —C₁₅H₃₁ >200 0.93 >200 0.66—C₁₆H₃₃ >200 0.85 77.8 0.9 —C₁₈H₃₇ N.D. N.D. 29 R² = coefficient ofdetermination

The foregoing biological data is also depicted in FIG. 1. Plasma levelsof bendamustine after administration of the foregoing esters aredepicted in FIG. 20.

Tumor Efficacy

Procedure for Nude Mouse MDA-MB-231 Tumor Efficacy Study:

Charles River Labs athymic nude mice were subcutaneous injected with5×10⁶ human tumor cells in matrigel. The tumor volume was monitoreduntil an average tumor size of ˜150 mm³ was obtained in the mousepopulation The mice were then randomized in to one of the nine treatmentgroups (summarized in table below) with a population of 10 mice pergroup (n=10). Each of the formulations were dosed at 100 μL fixed volumevia tail vein injection on day 1 and day 2 of the study. Mice wereweighed and the tumor volume measured every 3-4 days for 3 weeks of thestudy duration. This data is depicted in Table 2 and FIG. 2.

TABLE 2 Summary of Tumor Efficacy Data Against MB-231 of Bendamustine(BM1), Bendamustine C₁₂ Ester, and Bendamustine C₁₄ Ester: % TumorInhibition, % Morbidity/Mortality, and Tumor and Plasma Levels. Levelsat 1 hour (ng/mL) % Tumor % Morbidity/ Ester BM1 mg/kg Inh. MortalityTumor Plasma Tumor Plasma Solution BM1 37.5 77 10/0 — — 558 906 C₁₂ 5584 40/0 425 0 1932 4110 C₁₂ 80 94  80/20 644 16 4400 11650 C₁₄ 80 9040/0 396 14 7 21 HSA C₁₂ 55 79 10/0 261 2 1845 6307 nanoparticle C₁₂ 8091  70/40 762 292 7320 18757 C₁₂ 100 97 100/70 1565 39 15050 23933 C₁₄80 86.5 60/0 10430 5727 5500 15110

Procedure for Nude Mouse H460 Tumor Efficacy Study:

H460 tumor cells (large cell lung cancer) were cultured in RPMI-1640medium containing 10% FBS and with 95% air and 5% CO₂ at 37° C. Whenreaching to 80-90% confluent, the cells were detached by 0.25%Trypsin-EDTA solution within 5-10 minutes, neutralized with freshcultured medium, and counted by a cell counter (Cellometer, Auto T4 byNexcelom). 2×10⁶ cells/100 ul in the mix of medium and Matrigel (1:1ratio) solution was injected into right back flank of each nu/nu mouse.The implanted mice were monitored and measured with electric calipers.The study started when the tumors reached ˜150 mm³ in size. The micewere measured and randomized into 9 groups with 10 mice in each groupper the below table:

Tumor Efficacy Dosing Groups Dose (mg/kg Free Compound Formulation BaseEq.) Vehicle Control Solution NA Bendamustine HCl TREANDA 37.5Bendamustine C₁₂ ester Solution 55 Bendamustine C₁₂ ester Solution 80Bendamustine C₁₂ ester Nanoparticle 55 Bendamustine C₁₂ esterNanoparticle 80 Bendamustine C₁₂ ester Nanoparticle 100 Bendamustine C₁₄ester Solution 80 Bendamustine C₁₄ ester Nanoparticle 80

The formulations were administrated as 100 μL dose volume through tailvein injection within 30 minutes after compounds were formulated. Allthe mice were weighed and tumors measured twice weekly. At the last dayof the study, plasma, tumor, lung, liver, spleen, left kidney, brain andlegs were collected and quickly frozen for further analysis two hourspost-dosing. The results are shown in Table 3.

TABLE 3 Summary of Tumor Efficacy Data Against H-460 of Bendamustine(BM1), Bendamustine C₁₂ Ester, and Bendamustine C₁₄ Ester: % TumorInhibition, % Morbidity/Mortality, and Tumor and Plasma Levels. Levelsat 1 hour (ng/mL) % Tumor % Morbidity/ Ester BM1 mg/kg Inh. MortalityTumor Plasma Tumor Plasma Solution BM1 37.5 41.5  0/0 — — 1472 1550 C₁₂55 66  20/20 163 12 3760 6943 C₁₂ 80 75 100/50 206 12 5180 13600 C₁₄ 8081 100/40 8570 32233 4650 24633 HSA C₁₂ 55 47 10/0 154 0 1761 4743nanoparticle C₁₂ 80 64 40/0 321 0 2997 8053 C₁₂ 100 70 50/0 437 2 356512386 C₁₄ 80 40 10/0 4570 3648 1872 4467

Pharmacokinetic Study Experimentals

Procedure for Nude Mouse Tumor Efficacy PK Group:

A portion of the tumor bearing mice were dosed as a satellite PK group.Each group was dosed as described for the tumor efficacy group, howeverthe PK group was euthanized and tissue samples collected at 1, 3 and 6hours post dosing on day 2. The tissues collected included blood, lung,liver and tumor. Samples were analyzed for bendamustine HCl and thecorresponding ester analogue as described in the LC-MS protocol section.

LC-MS Experimental Protocol for PK Studies:

Plasma and other tissues were prepared for high performance liquidchromatography (HPLC)/mass spectrometric analysis according to astandard protocol following protein precipitation with acetonitrilecontaining an internal standard. The samples were then analyzed for bothbendamustine HCl and bendamustine esters of the invention and alprenolol(internal standard) via HPLC coupled with tandem mass spectrometry.Tissue samples were homogenized in sodium phosphate buffer and the valueobtained from the assay was multiplied by 3 to correct for dilutionduring processing.

Animal Dosing Protocol for PK Studies:

Adult animals (Charles River, Kingston, N.Y.; n=3 or 4/time point) wereused in all experiments. The mice or rats were not fasted overnightprior to IV dose administration via the lateral tail vein. IV doses wereadministered in a fixed dose volume of 100 μL in mice or a dose volumeof 1 mL/kg in rats. The mice were sacrificed by decapitation and trunkblood was collected into heparinized tubes at the sampling timesstipulated. For blood collection, each rat (unanesthetized) was placedin a clear Plexiglas® restraining tube, and blood samples (approximately0.25 mL) were drawn from a lateral tail vein into heparinized collectiontubes at the sampling times stipulated. (Note: No pre-dose samples wereobtained.) The exception to this procedure was the last sampling time inwhich the rats were sacrificed by decapitation and trunk blood wasobtained rather than blood via a tail vein. The blood samples wereplaced on wet ice until centrifuged to separate plasma. The plasmafraction was transferred into clean, dry tubes, frozen on dry ice andstored at approximately −20° C. pending analysis. Whole brains and otherhighly perfused organs (liver, lung, spleen, kidney and heart) wererapidly removed at the predetermined time points and frozen on dry ice.All tissue samples were also stored at approximately −20° C. pendinganalysis.

Pharmacokinetic Analysis:

The plasma concentration data for all mice and rats were entered intoExcel spreadsheets in preparation for pharmacokinetic analysis. Meanpharmacokinetic parameters were estimated by non-compartmental analysis(Gibaldi and Perrier 1982) of the plasma concentration versus time datausing WinNonlin software (Professional Version 4.1, PharsightCorporation, Palo Alto, Calif.). The terminal rate constant forelimination from plasma (β) was estimated by linear regression of theterminal portion of the semi-logarithmic plasma concentration versustime curve. The apparent terminal half-life (t1/2) was calculated as0.693 divided by β. The area under the plasma concentration versus timecurve from time zero to the time of the last measurable concentration(AUC0-t) after a single dose was determined by the linear trapezoidalrule. The area from zero to infinity (AUC0-∞) was calculated as the sumof AUC0-t and the area extrapolated from the last measurableconcentration to infinity (Clast/β). Concentrations pre-dose were allassumed to be zero for the purpose of calculation of the AUC. Anyconcentration that was below the limit of quantification (BLQ) after thelast quantifiable sampling time was considered to be an empty value forthe purpose of calculation of the AUC; it was treated as zero for thecalculation of the mean concentration for a given sampling time.

Data from the pharmacokinetic studies is depicted in Tables 4-17.

TABLE 4 Plasma Liquid Formulation Nanoparticle Formulation BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 5.7 ND 6.0 ND AUC_(0-t),66885 ND 55296 ND ng*h/mL AUC_(0-∞), 67131 ND 55417 ND ng*h/mL

The data from Table 4 is also depicted in FIG. 8.

TABLE 5 Blood Liquid Formulation Nanoparticle Formulation BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 6.3 ND 5.7 ND AUC_(0-t),91549 4424 58021 951 ng*h/mL AUC_(0-∞), 91856 ND 58112 ND ng*h/mL

The data from Table 5 is also depicted in FIG. 9.

TABLE 6 Brain Liquid Formulation Nanoparticle Formulation BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h ND ND ND ND AUC_(0-t), 9284299 1010 149 ng*h/mL AUC_(0-∞), ND ND ND ND ng*h/mL

The data from Table 6 is also depicted in FIG. 10.

TABLE 7 Liver Liquid Formulation Nanoparticle Formulation BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 4.2 0.8 2.6 4.5 AUC_(0-t),13901 2564 43785 13355 ng*h/mL AUC_(0-∞), 14111 2586 44713 13586 ng*h/mL

The data from Table 7 is also depicted in FIG. 11.

TABLE 8 Lung Liquid Formulation Nanoparticle Formulation BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 2.0 7.6 5.1 8.5 AUC_(0-t),22229 12619 15075 28327 ng*h/mL AUC_(0-∞), 22286 13246 15590 32297ng*h/mL

The data from Table 8 is also depicted in FIG. 12.

TABLE 9 Spleen Liquid Formulation Nanoparticle Formulation BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 2.3 3.1 1.6 5.4 AUC_(0-t),10201 25874 2598 13111 ng*h/mL AUC_(0-∞), 10362 25927 2735 13578 ng*h/mL

The data from Table 9 is also depicted in FIG. 13.

TABLE 10 Kidney Liquid Formulation Nanoparticle Formulation BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 6.4 5.3 7.0 4.9 AUC_(0-t),19489 2383 9665 1766 ng*h/mL AUC_(0-∞), 19966 2605 10725 1802 ng*h/mL

The data from Table 10 is also depicted in FIG. 14.

TABLE 11 Plasma, Blood, and Organ Levels of Bendamustine in Rat AfterAdministration of Bendamustine C₁₂ Ester Liquid Formulation, 30 mg/mL, 1mL/kg FIG. 15 Plasma Blood Brain Liver Lung Spleen Kidney t_(1/2), h 5.76.3 ND 4.2 2.0 2.3 6.4 AUC_(0-t), ng*h/mL 66885 91549 9284 13901 2222910201 19489 AUC_(0-∞), ng*h/mL 67131 91856 ND 14111 22286 10362 19966

The data from Table 11 is also depicted in FIG. 15.

TABLE 12 Plasma, Blood, and Organ Levels of Bendamustine C₁₂ Ester AfterAdministration of Bendamustine C₁₂ Ester Liquid Formulation, 30 mg/mL, 1mL/kg FIG. 16 Plasma Blood Brain Liver Lung Spleen Kidney t_(1/2), h NDND ND 0.8 7.6 3.1 5.3 AUC_(0-t), ng*h/mL ND 4424 299 2564 12619 258742383 AUC_(0-∞), ng*h/mL ND ND ND 2586 13246 25927 2605

The data from Table 12 is also depicted in FIG. 16.

TABLE 13 Plasma, Blood, and Organ Levels of Bendamustine in Rat AfterAdministration of Bendamustine C₁₂ Ester Nanoparticle Formulation, 30mg/mL, 1 mL/kg FIG. 17 Plasma Blood Brain Liver Lung Spleen Kidneyt_(1/2), h 6.0 5.7 ND 2.6 5.1 1.6 7.0 AUC_(0-t), ng*h/mL 55296 580211010 43785 15075 2598 9665 AUC_(0-∞), ng*h/mL 55417 58112 ND 44713 155902735 10725

The data from Table 13 is also depicted in FIG. 17.

TABLE 14 Plasma, Blood, and Organ Levels of Bendamustine C₁₂ Ester AfterAdministration of Bendamustine C₁₂ Ester Nanoparticle Formulation, 30mg/mL, 1 mL/kg FIG. 18 Plasma Blood Brain Liver Lung Spleen Kidneyt_(1/2), h ND ND ND 4.5 8.5 5.4 4.9 AUC_(0-t), ng*h/mL ND 951 149 1335528327 13111 1766 AUC_(0-∞), ng*h/mL ND ND ND 13586 32297 13578 1802

The data from Table 14 is also depicted in FIG. 18.

TABLE 15 Plasma Levels of Bendamustine in Rat After Dosing BendamustineC₁₂ Ester Nanoparticles at 3 mg-eq/kg, i.v.: Comparison of DifferentFormulations FIG. 19 Formulation Treanda HSA HSA/PLGA PLGA/PELA HSAw/PEG t_(1/2), h 0.16 ± 0.01  0.63 ± 0.09  0.41 ± 0.04  2.1 ± 0.3  1.6 ±0.3 AUC_(0-t), ng*h/mL 1609 ± 77  773 ± 58 1120 ± 113 856 ± 93 1194 ±186 AUC_(0-∞), ng*h/mL 1631 ± 81  781 ± 58 1132 ± 115 959 ± 92 1250 ±203 Vd, L/kg 0.42 ± 0.04  3.6 ± 0.7  1.6 ± 0.2 10.0 ± 2.2  6.1 ± 1.8 CL,mL/min/kg 31 ± 2  65 ± 5 46 ± 4 54 ± 5 44 ± 7 Mean ± SD, n = 4Bendamustine C12 Ester Nanoparticles

The data from Table 15 is also depicted in FIG. 5.

TABLE 16 Plasma Levels of Bendamustine C₁₂ Ester in Rat After DosingBendamustine C₁₂ Ester Nanoparticles at 3 mg-eq/kg, i.v.: Comparison ofDifferent Formulations FIG. 20 Formulation HSA HSA/PLGA PLGA/PELA HSAw/PEG t_(1/2), h ND 0.12 ± 0.01  2.3 ± 0.0  0.20 ± 0.01 AUC_(0-t),ng*h/mL 21 ± 2 18 ± 5  17 ± 3 55 ± 4 AUC_(0-∞), ng*h/mL ND 21 ± 7  36 ±5 56 ± 4 Vd, L/kg ND 47 ± 15 428 ± 59 23.3 ± 2.5 CL, mL/min/kg ND 4194 ±1071 2109 ± 252 1335 ± 99  Mean ± SD, n = 4

The data from Table 16 is also depicted in FIG. 6.

TABLE 17 Plasma Levels of Bendamustine C₁₆ Ester, Cyclohexyl Ester, and5-Decanyl Ester in Rat After Dosing Solution Formulations at 3 mg-eq/kgi.v. Bendamustine Ester Bendamustine Ester Bendamustine Ester Plasma C16bendamustine ester 5-decanyl ester Cy-hexyl bendamustine ester t_(1/2),h 0.46 0.28 0.16 0.20 AUC_(0-t), ng*h/mL 505 1208 633 504 AUC_(0-∞),ng*h/mL 617 1214 642 All < MQL 517 All < MQL Vd, L/kg 3.3 1.0 1.1 1.6CL, mL/min/kg 82 41 78 96 1.85% solvent 1.6% solvent 1.4% solvent Mean,n = 3 Diluted into saline from 1/1/1 DMA/PG/Solutol

Another embodiment of the invention, bendamustine C₁₄ ester wasformulated into a nanoparticle intraveneous formulation according to themethods described above and administered to CD-1 mice. The amount ofbendamustine (BM1) and bendamustine C₁₄ ester was determined in the miceplasma. The results of these experiments are summarized in Table 18.

TABLE 18 BM1 C₁₄ Ester BM1 C₁₄ Ester BM1 C₁₄ Ester Plasma 30 mg-eq/kg 55mg-eq/kg 80 mg-eq/kg t_(1/2), h 1.17 1.52 0.92 0.98 0.95 1.09 AUC₀₋₆,ng*h/mL 4507 31693 4152 48934 6007 46335 AUC_(0-∞), ng*h/mL 4524 317414188 49020 6068 46485 Vd, L/kg ND 3.2 ND 2.5 ND 5.2 CL, mL/min/kg ND 24ND 29 ND 56 Mean, n = 3 Albumin Nanoparticle

PEG-ylated esters of bendamustine were also tested. Data for PEG-2000and PEG-5000 esters of bendamustine is depicted in Tables 19 and 20below. This data is also depicted in FIG. 19.

TABLE 19 Plasma Levels of Bendamustine in Rat Dased as BendamustinePEG-2000 Ester, 3 mg-eq/kg i.v., 1 mL/kg Bendamustine Rat 1 Rat 2 Rat 3Rat 4 Mean st. dev. sem t_(1/2), h 0.46 0.23 0.50 0.22 0.36 0.15 0.07AUC₀₋₆, ng*h/mL 2100 2866 1494 1379 1960 682 341 AUC_(0-∞), ng*h/mL 21092868 1506 1382 1966 680 340 Vd, L/kg 0.95 0.35 1.46 0.70 0.87 0.46 0.23CL, mL/min/kg 24 17 33 36 28 9 4 bendamustine PEG-2000 ester 3 mg-eq/kg,1 mL/kg

TABLE 20 Plasma Levels of Bendamustine in Rat Dased as BendamustinePEG-5000 Ester, 3 mg-eq/kg i.v., 1 mL/kg Bendamustine Rat 1 Rat 2 Rat 3Rat 4 Mean st. dev. sem t_(1/2), h 0.16 0.48 0.47 0.48 0.40 0.16 0.08AUC₀₋₆, ng*h/mL 808 1735 1572 1050 1291 435 217 AUC_(0-∞), ng*h/mL 8171741 1579 1058 1299 434 217 Vd, L/kg 0.84 1.19 1.29 1.99 1.32 0.48 0.24CL, mL/min/kg 61 29 32 48 42 15 8 bendamustine PEG-5000 ester 3mg-eq/kg, 1 mL/kg

Analysis of the In-Vitro Stability of Bendamustine Esters

Tumor S9 Preparation:

Charles River Labs athymic nude mice bearing breast (MB-231) ornon-small-cell lung cancers (H460) were sacrificed. Tumors wereimmediately removed and rinsed with ice-cold 1.15% KCl. The tumors wereweighed, cut and minced. Minced tissues were mixed with 4× (v/w)ice-cold SET buffer (250 mM sucrose, 5.4 mM Na₂EDTA and 20 mM Tris, pH7.4) and homogenized with tissue homogenizers. Homogenates weretransferred into clean polycarbonate ultracentrifuge tubes and spun at10,000 g at 4° C. for 20 min. Lipid at the top of the ultracentrifugetubes was removed with cotton swabs, and the supernatant (S9) aliquotswere stored in a −80° C. freezer.

In Vitro Incubation:

Incubation mixture containing 50 mM phosphate buffer (pH 7.4), anNADPH—(reduced nicotinamide adenosine diphosphate) regenerating systemand 1 mg/mL tumor S9 were pre-warmed in a 37° C. water bath. Reactionswere initiated by adding 1 μL, of bendamustine C6, C8, C12 or C14 esterinto separate incubation mixtures to obtain final concentrations of eachbendamustine ester of 10 μM. At designed time points, 100-μL aliquots ofthe incubation mixtures were removed and mixed with 400 μL of stopsolution (4 or 80 μM tiagabine [IS] in a solution of 0.1% formicacid/acetonitrile). All samples were vortex-mixed and placed on ice forat least 10 min and then the protein was precipitated by centrifuging inan Eppendorf 5417R centrifuge at 14000 rpm×8 min. The supernatant wastransferred into HPLC vials and 10 μL was injected for analysis usinghigh performance liquid chromatography with tandem mass spectrometricdetection.

LC-MS/MS Method:

The LC-MS/MS system consisted of a Shimadzu HPLC and a Sciex API 4000MS. The chromatography was performed on a Phenomenex 00B-4448-B0, LunaPFP (2) column (50×2 mm, 5 μm particle size). The total mobile phaseflow rate was 0.5 mL/min. The gradient began at 70% mobile phase A (0.1%aqueous trifluoroacetic acid) and 30% mobile phase B (100%acetonitrile). The proportion of mobile phase B was then linearlyincreased to 95% within 0.5 min and was maintained at that ratio for 1.3min, re-equilibrating to initial conditions within 1 min. The massspectrometer was tuned to the respective optimal conditions for eachbendamustine ester, monitoring transitions of 442.2/340.1 (C6),470.2/340.1 (C8), 526.3/340.1 (C12) and 554.3/340.1 (C14).

Data from the in-vitro stability studies is set forth in FIGS. 3 and 4.

Electron Microscopy Experimentals

Sample Preparation for c-TEM Study:

Sample was solubilized by adding 7.8 mL of water for injection (WFI) tothe sample and mixed by inverting by hand. Sample dissolved quickly,˜2-5 minutes, with no visible undissolved particles in the solution. Thesample was preserved in vitrified ice supported by carbon coated holeycarbon films on 400 mesh copper grids. The sample was prepared byapplying a 3 μL drop of undiluted sample solution to a cleaned grid,blotting away with filter paper and immediately proceeding withvitrification in liquid ethane. Grids were stored under liquid Nitrogenuntil transferred to the electron microscope for imaging.

c-TEM Imaging Parameters:

Electron microscopy was performed using an FEI Tecnai T12 electronmicroscope, operating at 120 KeV equipped with an FEI Eagle 4K×4K CCDcamera. The grid was transferred into the electron microscope using acryostage that maintains grids at a temperature below −170 C. Images ofthe grid were acquired at multiple scales to assess the overalldistribution of the specimen. After identifying potentially suitabletarget areas for imaging at lower magnifications, high magnificationimages were acquired at nominal magnifications of 52,000× (0.21nm/pixel), and 21,000× (0.50 nm/pixel). The images were acquired at anominal underfocus of −4 μm (52,000×) and −5 μm (21,000×) and electrondoses of ˜10-15 e/Å2.

Results of the electron microscopy experiments is depicted in FIG. 7.

Cross-Linking Experiments with HSA Nanoparticles Formulation ofBendamustine Esters

Circulation times of bendamustine and bendamustine esters of theinvention can be extended using HSA-based nanoparticle formulationwherein the protein moieties are covalently cross-linked after thenanoparticle structures are formed. See, e.g., K. Langer et al.International Journal of Pharmaceutics 347 (2008) 109-117. This wouldprovide more structure to the surface coating and would prevent a rapidrelease of the nanoparticle contents. This could also provide “stealth”protection of the nanoparticle by introducing a PEG group to thecross-linking agent. Effective encapsulation and hardening of the HSAnanoparticle was demonstrated using the commercially availabledialdehyde, glutaraldehyde. Introduction of an appropriately-sized PEGmoiety could be added using, for example, the trifunctional PEGcross-linking agent prepared as shown below:

Procedure:

HSA nanoparticles were diluted with DI water to a concentration of 1.5mg/mL C14 ester of bendamustine, which corresponds to a 6 mg/mLconcentration of HSA. The resulting suspension of nanoparticles was thenaliquoted in 1 mL portion into five glass vials outfitted with amagnetic stir bar. The appropriate amount of a 50% glutaraldehydesolution was added and each vial was capped and stirred at roomtemperature over-night. Each sample was then diluted 1 to 10 intoN-methylpyrrolidone (NMP) and the sample spun for ˜2 minutes using amicro-centrifuge to remove HSA and cross-linked HSA nanoparticles. Thesupernatant was then analyzed by HPLC and the concentration (peak area)of the C14 ester of bendamustine was determined to confirm particleencapsulation. The table below shows the concentration ofun-encapsulated C14 ester of bendamustine as a function of the μL ofgluturaldehyde:

μL of glutaraldehyde HPLC Peak Area 0 4882.02 2 4788.99 5 4778.11 104714.02 20 29.55

The data shows that the addition of glutaraldehyde at a ratio of 3.33μL/mg of HSA was suitable to result in a system of cross-linked,bendamustine ester-containing nanoparticles.

In Vivo Multiple Myeloma Model Materials and Methods

RPMI 8226 (Human Plasmacytoma, Myeloma B Cells) ATCC # CCL-155;

ECM Gel (Matrigel), Sigma-Aldrich, Cat # E1270, 5 ml

RPMI (Beit Haemek, Lot: 1110235)

Velcade® (Bortezomib) 3.5 mg lyophilized in vial, Lot# BIZSC00

Bendamustine (Lot # TD-D0815, API Lot #00039P0012)

Bendamustine C12 ester nanoparticles (C12NP), Lot #2861-242-22, 17.6mg/vial

Sodium chloride

Water for injections (DEMO S.A.)

Test Animals

80 CB.17 SCID female mice, 4-6 weeks old, 16-20 grams, obtained fromHarlan animal breeding center

Cells Preparation

Cells (originated from ATCC) were cultured on RPMI medium. Cellsuspension was centrifuged and resuspended in 50% Matrigel/HBSS to afinal concentration of 7×10⁷ cells/ml. The suspension was implanted s.c.in the right flank of the anesthetized mouse at a volume of 100 μl.

Compounds Preparation

VELCADE® was prepared once a week. Seven ml saline were added to theoriginal vial containing 3.5 mg powder resulting in 0.5 mg/ml. Three mlof this solution were added to 27 ml saline to receive 0.05 mg/mlconcentration.

bendamustine preparation: 13.5 mg was dissolved in 3.6 ml of 1:1 mixtureof 0.9% saline/5% mannitol just before the treatment. 1.05 ml of thissolution was added to 0.95 ml diluent for 2 mg/ml solution.

C12NP preparation: 3.1 ml SWFI was added into sample vial containing17.62 mg just before the treatment. 1.05 ml of this solution was addedto 0.95 ml diluent for 2 mg/ml solution.

Experimental Design

Mice were implanted subcutaneously, with 7×106 RPMI 8226 cells/mouse (in50% Martigel/HBSS) on Day 0. On day 21, mice were sorted by the optimalaverage tumor volume (˜150 mm³) and were allocated into eight groups of9 mice each.

Gr. N Agent Route Dose & schedule 1 9 WFI iv Days 1 & 2 2 9 Velcade iv0.5 mg/kg biweekly 3 9 Bendamustine iv 20 mg/kg on days 1&2 4 9Bendamustine iv 37.5 mg/kg on days 1&2 5 9 C12NP iv 20 mg/kg on days 1&26 9 C12NP iv 37.5 mg/kg on days 1&2

It should be noted that the nature of SCID mice (i.e., severelyimmune-compromised) make them more fragile/sensitive animals, and theyare therefore less able to tolerate the doses of C12NP used in NUDE mice(which are comparatively less immune-compromised). Specifically, dosingSCID mice at 100 mg/kg and 70 mg/kg revealed unacceptable toleranceissues (i.e., more than 20% body weight loss)(data not shown). It isbelieved that this (tolerance difference between nude vs. SCID) is astrain-related phenomenon. As such, the study proceeding using doses of37.5 mg/kg and 20 mg/kg of C12NP.

Statistical Analysis

Tumor volume was calculated as follows:

$\pi \times ( \frac{width}{2} )^{2} \times {{length}.}$

The analysis of weight gain and tumor volume progression was made usingone-way ANOVA followed by Tukey post-hoc comparisons.

Results

The treatment responses are summarized in FIGS. 23 and 24. All thetreatments except C12NP 20 mg/kg significantly inhibited tumor growthcompared to control group (see data in Table 21). At 37.5 mg/kg, theefficacy of Bendamustine and C12NP were similar, with 81% and 70%inhibition of tumor growth. However, as can be seen in FIG. 24, thetolerability of C12NP was better than Bendamustine as measured by lessweight loss in animals treated with C12NP.

TABLE 21 Summary of Results (day 46) mean tumor Max BW volume, No. ofNo. of (mean) No. of No. og Compound Regimen (mean ± se) % TGI PR CRreduction TRD nTRD 1. Vehicle WFI iv on 597 ± 71 days 1 and 2 2.Velcade ® i.v. biwk 226 ± 35 62*** 0 0 −0.4% 0 0 0.5 mg/kg day 20 3.Bendamustine iv on days 1 205 ± 44 66*** 0 0 −2.7% 0 0 20 mg/kg and 2day 22 4. Bendamustine iv on days 1 113 ± 23 81*** 0 0 −6.2% 0 0 37.5mg/kg and 2 day 25 5. C12NP iv on days 1 408 ± 49 32   0 0 −1.5% 0 0 20mg/kg and 2 day 25 6. C12NP iv on days 1 180 ± 31 70*** 0 0 −3.1% 0 037.5 mg/kg and 2 day 25 ***p < 0.001 (one-way Anova, Tukey post-hoctest)

What is claimed:
 1. A compound of formula I:

wherein R₁ is C₆-C₂₄alkyl or polyethylene glycol.
 2. The compound ofclaim 1, wherein R₁ is C₈-C₂₄alkyl.
 3. The compound of claim 1, whereinR₁ is C₁₀-C₂₄alkyl.
 4. The compound of claim 1, wherein R₁ isC₁₂-C₂₄alkyl.
 5. The compound of claim 1, wherein R₁ is C₁₄-C₂₄alkyl. 6.The compound of claim 1, wherein R₁ is C₁₆-C₂₄alkyl.
 7. The compound ofclaim 1, wherein R₁ is C₁₈-C₂₄alkyl.
 8. The compound of claim 1, whereinR₁ is C₁₀alkyl.
 9. The compound of claim 1, wherein R₁ is C₁₂alkyl. 10.The compound of claim 1, wherein R₁ is C₁₄alkyl.
 11. The compound ofclaim 1, wherein R₁ is C₁₆alkyl.
 12. The compound of claim 1, wherein R₁is C₆alkyl.
 13. The compound of claim 1, wherein the compound of formulaI is


14. The compound of claim 1, wherein the compound of formula I is


15. The compound of claim 1, wherein the compound of formula I is


16. The compound of claim 1, wherein the compound of formula I is


17. A pharmaceutical composition comprising a compound according toformula IA:

wherein R is C₁-C₂₄alkyl or polyethylene glycol; and a pharmaceuticallyacceptable carrier or diluent.
 18. The pharmaceutical composition ofclaim 17, wherein R is C₁₀-C₂₄alkyl
 19. The pharmaceutical compositionof claim 17, wherein R is C₁₀alkyl.
 20. The pharmaceutical compositionof claim 17, wherein R is C₁₂alkyl.
 21. The pharmaceutical compositionof claim 17, wherein R is C₁₄alkyl.
 22. The pharmaceutical compositionof claim 17, wherein R is C₁₆alkyl.
 23. Nanoparticles comprising acompound according to formula IA:

wherein R is C₁-C₂₄alkyl or polyethylene glycol.
 24. A method oftreating cancer in a patient comprising administering to the patient acompound according to formula IA:

wherein R is C₁-C₂₄alkyl or polyethylene glycol.
 25. The method of claim24, wherein the cancer is chronic lymphocytic leukemia, Hodgkin'sdisease, indolent non-Hodgkin's lymphoma, aggressive non-Hodgkin'slymphoma, multiple myeloma, acute lymphocytic leukemia, breast cancer orlung cancer.
 26. The method of claim 24, wherein the cancer is selectedfrom sarcoma, bladder cancer, cervical cancer, testicular cancer,melanoma, glioblastoma, colon cancer, head and neck cancer, ovariancancer, and prostate cancer.
 27. The method of any one of claims 24 to26, wherein the patient is resistant to one or more chemotherapeuticagents.
 28. The method of claim 27, wherein the one or morechemotherapeutic agents is an alkylating agent.
 29. A compound offormula II:

wherein R₂ is C₁-C₂₄alkylene; and R₃ is —COOC₁₋₃alkyl; or R₂-R₃ isC₁-C₂₄alkyl; or a pharmaceutically acceptable salt form thereof.
 30. Thecompound of claim 29, wherein R₂-R₃ is C₈-C₂₄alkyl.
 31. The compound ofclaim 29, wherein R₂-R₃ is C₁₀-C₂₄alkyl.
 32. The compound of claim 29,wherein R₂-R₃ is C₁₂-C₂₄alkyl.
 33. The compound of claim 29, whereinR₂-R₃ is C₁₄-C₂₄alkyl.
 34. The compound of claim 29, wherein R₂-R₃ isC₁₆-C₂₄alkyl.
 35. The compound of claim 29, wherein R₂-R₃ isC₁₈-C₂₄alkyl.
 36. The compound of claim 29, wherein R₂-R₃ is C₁₀alkyl.37. The compound of claim 29, wherein R₂-R₃ is C₁₂alkyl.
 38. Thecompound of claim 29, wherein R₂-R₃ is C₁₄alkyl.
 39. The compound ofclaim 29, wherein R₂-R₃ is C₁₆alkyl.
 40. The compound of claim 29,wherein R₂ is C₂alkyene and R₃ is —COOCH₃.
 41. The compound of claim 29,wherein the compound of formula II is


42. The compound of claim 29, wherein the compound of formula II is


43. The compound of claim 29, wherein the compound of formula II is


44. The compound of claim 29, wherein the compound of formula II is


45. A pharmaceutical composition comprising a compound according toclaim 29 and a pharmaceutically acceptable carrier or diluent. 46.Nanoparticles comprising a compound according to claim
 29. 47. A methodof treating cancer in a patient comprising administering to the patienta compound according to claim
 29. 48. The method of claim 47, whereinthe cancer is chronic lymphocytic leukemia, Hodgkin's disease, indolentnon-Hodgkin's lymphoma, aggressive non-Hodgkin's lymphoma, multiplemyeloma, acute lymphocytic leukemia, breast cancer or lung cancer. 49.The method of claim 47, wherein the cancer is selected from sarcoma,bladder cancer, cervical cancer, testicular cancer, melanoma,glioblastoma, colon cancer, head and neck cancer, ovarian cancer, andprostate cancer.
 50. The method of any one of claims 47 to 49, whereinthe patient is resistant to one or more chemotherapeutic agents.
 51. Themethod of claim 50, wherein the one or more chemotherapeutic agents isan alkylating agent.
 52. Nanoparticles comprising a compound accordingto any one of claims 1 to
 16. 53. The nanoparticles of claim 52, furthercomprising human serum albumin.
 54. The nanoparticles of claim 52 or 53,further comprising poly-lactic glycolic acid.
 55. The nanoparticles ofclaim 52 or 53, further comprising a polyethylene glycol.
 56. Thenanoparticles of claim 52 or 53, further comprising a poly-lacticglycolic Acid (PLGA) and polyoxyethylene lactic acid copolymer.
 57. Thenanoparticles of claim 53, wherein the human serum albumin iscross-linked with a dialdehyde.
 58. The nanoparticles of claim 57,wherein the dialdehyde is glutaraldehyde.
 59. A lyophilized compositioncomprising nanoparticles according to any one of claims 52 to
 58. 60.The lyophilized composition of claim 59, further comprising one or moreof mannitol, trehalose, sucrose, or NaCl.
 61. A method of treatingcancer in a patient comprising administering to the patient thenanoparticles according to any one of claims 52 to
 58. 62. The method ofclaim 61, wherein the cancer is chronic lymphocytic leukemia, Hodgkin'sdisease, indolent non-Hodgkin's lymphoma, aggressive non-Hodgkin'slymphoma, multiple myeloma, acute lymphocytic leukemia, breast cancer orlung cancer.
 63. The method of claim 61, wherein the cancer is selectedfrom sarcoma, bladder cancer, cervical cancer, testicular cancer,melanoma, glioblastoma, colon cancer, head and neck cancer, ovariancancer, and prostate cancer.
 64. The method of any one of claims 61 to63, wherein the patient is resistant to one or more chemotherapeuticagents.
 65. The method of claim 64, wherein the one or morechemotherapeutic agents is an alkylating agent.